UNITED STATES OF AMERICA
TO ALL WHOM IT MAY CONCERN:
BE IT KNOWN THAT WE:
Gregory R. J. THATCHER, 371 Alfred Street, Kingston, Ontario, K7K4H6, Canada, citizen of
United Kingdom,
and
Brian M. BENNETT, 36 Fairway Hill Crescent, Kingston, Ontario, K7M 2B4, Canada, citizen of
Canada,
and
James N. REYNOLDS, 52 Herchmer Crescent, Kingston, Ontario, K7M 2V9, Canada, citizen of
Canada,
and
Khem JHAMANDAS, 17 Jorene Drive, Kingston, Ontario, K7M 3X5, Canada, citizen of Canada
have invented:
METHOD OF MITIGATING PAIN
of which the following is a specification:
1
METHODS AND COMPOSITIONS FOR MITIGATING PAIN
FIELD OF THE INVENTION
This invention relates to nitrate esters and use thereof in mitigating pain and effecting
analgesia. More particularly this invention relates to organic nitrates which have therapeutic
utility as analgesics, anti-inflammatory agents and sedatives.
BACKGROUND OF THE INVENTION
The nitrate ester, glyceryl trinitrate (GTN), or nitroglycerin, has been used as a
vasodilator in the treatment of angina pectoris for over a hundred years, and the dominant,
contemporary belief is that GTN exerts its therapeutic effect through in vivo release of nitric
oxide (NO). Other organic nitrates (nitrate esters), such as isosorbide dinitrate, have also been
identified as effective and clinically important vasodilators. NO itself has been identified as
Endothelium Derived Relaxing Factor (EDRF) and several classes of compounds, for example
nitrosothiols, in addition to organic nitrates, have been proposed as NO donors or NO
prodrugs.
Several organic nitrates, in which an alkyl mononitrate is appended to a moiety with
analgesic properties, such as aspirin (ASA) or a Non-Steroidal Anti-Inflammatory Drug
(NSAID) have been reported as analgesics which possess reduced gastro-intestinal irritation
and ulceration properties, purportedly through release of NO. The combination of the
vasodilator nitroglycerin with opioid analgesics such as morphine, has been suggested to be
effective in the management of both surgical and cancer pain. However, no attempt has been
made to develop organic nitrates themselves as analgesic agents, that is, organic nitrates that do
not rely on an ASA or NSAID moiety, nor an opiate, for analgesic properties. Thus, there is
a need for synthetic organic nitrates as new and useful therapeutic agents for treatment and
mitigation of pain associated with disease states and chemotherapy of those disease states.
OBJECT OF THE INVENTION
2
It is an object of the present invention to provide methods and compositions for use in
treating pain and/or conditions associated with pain. Another object of the present invention
is to provide methods and compositions for providing analgesia and/ or sedation.
5 BRIEF DESCRIPTION OF THE INVENTION
The present invention is based, at least in part, on the recognition that, although the
potent vasodilatator effects of organic nitrates may be either (a) deleterious to or,
alternatively, (b) synergistic with their analgesic effects, regulation of these two effects is
required for the development of therapeutic agents useful in treatment and mitigation of pain.
10 Pain may be treated or mitigated by, for example, an analgesic, anti-inflammatory and/ or
□ sedative agent.
2 Possible deleterious effects of organic nitrates may arise, for example, through an NO-
:~1 donor potentiating hyperalgesia via a cyclic guanosine-3,5-monophosphate (cGMP)-
^ independent mechanism. Alternatively, synergistic effects of organic nitrates may arise, for
iiJ15 example, through the ability of an NO-donor to induce analgesia by activation of soluble
U guanylyl cyclase (GCase) and elevation of cGMP levels. The present invention relates to
:^ methods for treating or mitigating pain through use of an organic nitrate, wherein regulation
i y
; fi of these two effects is achieved. According to the invention, selection of an appropriate
;| organic nitrate provides modulation and balance between the ability of the organic nitrate to
20 release NO and its potency for GCase activation. Inasmuch as gastrointestinal toxicity is
known to be a deleterious side effect of some analgesic drugs and that NO donor molecules
are gastro-protective, it is set forth herein that therapeutic analgesia can be achieved through
utilization of an appropriate organic nitrate. This statement is based, at least in part, on
bioassay data on such compounds.
25 This invention provides methods and compositions which are useful in treating pain,
inhibiting inflammation, and/or providing analgesia. Methods of the invention involve
administering to a subject a therapeutic compound (nitrate ester) which provides analgesia.
The methods and compositions of the invention are useful for the treatment and mitigation of
pain associated with disorders and disease states and chemotherapy of those disease states. The
methods and compositions of the invention can be used therapeutically to treat acute, chronic
and/or inflammatory pain in conditions such as, but not limited to, nerve injury, post-
herpetic neuralgia, arthritis, diabetic neuropathy, dysmenorrhea, endometriosis, phantom
limb pain, pain associated with cancer and post-operative pain, or can be used prophylactically
in a subject susceptible or predisposed to these conditions. In certain preferred embodiments,
a therapeutic compound used in the method of the invention interacts with guanylyl cyclase,
effecting analgesia. In other preferred embodiments, a therapeutic compound used in the
method of the invention modulates levels of the cyclic nucleotides cyclic guanosine-3',5'-
monophosphate (cGMP) and cyclic adenosine-3',5'- monophosphate (cAMP).
In one aspect, the invention provides a method for treating pain, treating or inhibiting
inflammation, providing analgesia, providing sedation, mitigating anxiety and/ or providing
anaesthesia in a subject, comprising administering to a subject in need thereof an effective
amount of a therapeutic compound, wherein the therapeutic compound is of the formula (la):
(la)
in which E, F„ F 2 , G 1( andlSsare the same or different organic radicals which may be
joined in cyclic ring systems, and which ihaj^contain inorganic counterions;
with the proviso tE&swhen E and G, are methylene groups and F, is H, G 2 is not a
■nN »7N
nitrate group, nor R -Z -;
wherein R N is any aryl or heteroaryl group and Z N is (CO^-X^^-Y*
wherein mm, nn, oo are 0 or 1 and X N ,Y N are NH, NR™, O or CH 2 ;
wherein R 1 ™ is a short chain alkyl group (C, - C 12 ).
oo»
4
In a preferred emB&diment, F 2 is a nitrate group and E, ¥ u G u G 2 are the same or
different organic radicals which m^b^|oined in cyclic ring systems, and which may contain
inorganic counterions;
with the proviscrthat^when E and G x are methylene groups and ¥ t is H, G 2 is not a
nitrate group, nor R N -Z N -;
wherein R N is any aryl or heteroaryl group and Z N is (CO) mm -X N nn -Y N 00 ;
wherein mm, nn, oo are 0 or 1 and X N ,Y N are NH, NR™, O or CH 2 ;
herein R m is'a^kQrtchain alkyl group (C x - C 12 ) r ^
In another aspect, the invention provides a method for treating pain, treating or
inhibiting inflammation, providing analgesia, providing sedation, mitigating anxiety and/ or
providing anaesthesia in a subject, comprising administering to a subject in need thereof an
effective amount of a therapeutic compound, wherein the therapeutic compound is of the
formula (lb):
(ib)
^^^^l in which F 2 is ah^ganic radical which may be joined in a cyclic ring system with G 2 ,
^^and which may contain inoWiic counterions; E and G x are both methylene groups; F l is H;
( andG 2 isR N -Z N -;
wherein R N is an organic radical possessing a heteroaryl group containing P or S atoms
where said P or S are positioned P, y, or 8 to a nitrate group as identified in formula I; and Z N
25 isW N mm -X N nn -Y N O0 ;
wherein mm, nn and oo are 0 or 1; and W N , X N , Y N are NH, NR™, CO, O or CH 2 ;
wherein R 1 ™ is a short chain alkyl group (C, - C 12 ).
In a preferred embodiments F 2 is a nitrate group; E and Gj are methylene groups; Fj is
H; and G 2 is R N -2 N -;
^7
ty
5
10
wherein R N is an organic radical possessing an heteroaryl group containing P or S
atoms where said P or S are positioned p, y, or 5 to a nitrate group as identified in formula I;
andZ N isW N mm -X N nn -Y N 00 ;
wherein mm, nn, oo are 0 or 1 and W N , X N , Y N are NH, NR™, CO, O or CH 2 ;
wherein is a short chain alkyl group (Cj - C 12 ).
In another aspect, the invention provides a method for treating pain, treating or
inhibiting inflammation, providing analgesia, providing sedation, mitigating anxiety and/ or
providing anaesthesia in a subject, comprising administering to a subject in need thereof an
effective amount of a therapeutic compound, wherein the therapeutic compound is of the
formula (Ic):
25
(Ic)
in wr
hichEis(R 1 R 2 C) m and^^CF 1 F 2 - is R l9 -(R 3 R 4 C) p -(R 17 R 18 C) n -;
wherein: m, n, p are integers from 0 to 10;
R 3,17 are each independently hydrogen, a nitrate group, or A; and
R 1,4 are each independently hydrogen, or A;
where A is selected from a substituted or unsubstituted aliphatic group (preferably a
branched or straight-chain aliphatic moiety having from 1 to 24 carbon atoms in the chain,
which optionally may contain O, S, NR 6 and unsaturations in the chain, optionally bearing
from 1 to 4 hydroxy, nitrate, amino, aryl, or heterocyclic groups; an unsubstituted or
substituted cyclic aliphatic moiety having from 3 to 7 carbon atoms in the aliphatic ring,
which optionally may contain O, S, NR 6 and unsaturations in the ring, optionally bearing
from 1 to 4 hydroxy, nitrate, amino, aryl, or heterocyclic groups; an unsubstituted or
substituted aliphatic moiety constituting a linkage of from 0 to 5 carbons, between R 1 and R 3
6
and/or between R 17 and R 4 , which optionally may contain O, S, NR 6 and unsaturations in the
linkage, and optionally bearing from 1 to 4 hydroxy, nitrate, amino, aryl, or heterocyclic
groups); a substituted or unsubstituted aliphatic group (preferably a branched, cyclic or
straight-chain aliphatic moiety having from 1 to 24 carbon atoms in the chain) containing
5 carbonyl linkages (e.g., C=0, C=S, C=NOH), which optionally may contain O, S, NR 6 and
unsaturations in the chain, optionally bearing from 1 to 4 hydroxy, nitrate, amino, aryl, or
heterocyclic groups; a substituted or unsubstituted aryl group; a heterocyclic group; amino
(including alkylamino, dialkylamino (including cyclic amino, diamino and triamino moieties),
arylamino, diarylamino, and alkylarylamino); hydroxy; alkoxy; a substituted or unsubstituted
10 aryloxy;
□ wherein X is F, Br, CI, N0 2 , CH 2 , CF 2 , 0, NH, NMe, CN, NHOH, N 2 H 3 , N 2 H 2 R 13 ,
1 N 2 HR 13 R 14 , N„ S, SCN, SCN 2 H 2 (R ,5 ) 2 , SCN 2 H 3 (R 15 ), SC(0)N(R ,5 ) 2 , SC(0)NHR 15 , S0 3 M,
^ SH, SR 7 , S0 2 M, S(0)R 8 , S(0) 2 R 9 , S(0)OR 8 , S(0) 2 OR 9 , P0 2 HM, P0 3 HM, P0 3 M 2 ,
P(0)(OR ,5 )(OR 16 ), P(0)(OR 16 )(OM), P(0)(R 15 )(OR 1 '), P(0)(OM)R 15 , C0 2 M, C0 2 H, C0 2 R u ,
m C(0), C(0)R 12 , C(0)(OR 13 ), P0 2 H, P0 2 M, P(0)(OR H ), P(0)(R 13 ), SO, S0 2 , C(0)(SR 13 ), SR 5 ,
U SSR 7 or SSR 5 ;
IS Y is F, Br, CI, CH 3 , CF 2 H, CF 3 , OH, NH 2 , NHR 6 , NR 6 R 7 , CN, NHOH, N 2 H 3 ,
! 5 N 2 H 2 R 13 , N 2 HR 13 R 14 , N„ S, SCN, SCN 2 H 2 (R 15 ) 2 , SCN 2 H 3 (R 15 ), SC(0)N(R 15 ) 2 , SC(0)NHR 15 ,
I S0 3 M, SH, SR 7 , S0 2 M, S(0)R 8 , S(0) 2 R 9 , S(0)OR 8 , S(0) 2 0R 9 , P0 2 HM, P0 3 M 2 ,
20 P(0)(0R 15 )(0R 16 ), P(0)(OR 16 )(OM), ?{0)(K ls ){OK\ P(0)(OM)R 15 , C0 2 M, C0 2 H, C0 2 R n ,
C(0)R 12 , C(0)(OR 13 ), C(0)(SR 13 ), SR 5 , SSR 7 or SSR 5 , or does not exist;
R 2 , R 5 , R 18 , R 19 are optionally hydrogen, A or X-Y;
R 6 , R 7 , R 8 R 9 , R", R 12 , R 13 , R 14 , R 15 , R 16 are the same or different alkyl or acyl groups
containing 1-24 carbon atoms which may contain 1-4 ON0 2 substituents; or Q - C 6
25 connections to R 1 - R 4 in cyclic derivatives which may contain 1-4 ON0 2 substituents; or are
each independently hydrogen a nitrate group or A;
M is H, Na + , K + , NH 4 + , N + H k R 11 (4 . k) where k is 0-3; or other pharmaceutical^
acceptable counterion;
and with the proviso that when m = n = p = 1 and R 19 , R 2 , R 18 , R 1 = H and R 17 , R 3
are nitrate groups, R 4 is not H.
In a preferred embodiment, R 19 is X-Y.
In other embodiments, R 1 and R 3 are the same or different and selected from H and C r
C 4> alkyl chains, which chains may include one O linking R'and R 3 to form pentosyl, hexosyl,
cyclopentyl, or cyclohexyl rings, which rings may optionally bear hydroxyl substituents;
R 2 and R 4 are the same or different and selected from H, a nitrate group, C r C 4 alkyl
chains optionally bearing 1-3 nitrate groups, and acyl groups (-C(0)R 5 );
R 7 , R 11 are the same or different Q - C 8 alkyl or acyl;
R 5 , R 6 , R 8 R 9 , R 12 , R 13 , R 14 , R 15 , R 16 are the same or different and are alkyl groups
containing 1-12 carbon atoms which may contain 1-4 ON0 2 substituents; or C, or C2
connections to R 1 - R 3 in cyclic derivatives; and
M is H, Na + , K + , NH 4 + or N^R'^where k is 0-3.
In other embodiments, m=l,n = 0, p=l.
In further embodiments, X is CH 2 , O, NH, NMe, CN, NHOH, N 2 H 3 , N 2 H 2 R 13 ,
N 2 HR"R 14 , N„ S, SCN, SCN 2 H 2 (R 15 ) 2 , SCN 2 H 3 (R 15 ), SC(0)N(R ,5 ) 2 , SC(0)NHR 15 , S0 3 M,
SH, SR 7 , S0 2 M, S(0)R s , S(0) 2 R 9 , S(0)OR 8 , S(0) 2 OR 9 , P0 3 HM, P0 3 M 2 , P(0)(OR 15 )(OR 16 ),
P(0)(OR 16 )(OM), P^fR'^OR 8 ), P(0)(OM)R 15 , C0 2 M, C0 2 H, C0 2 R U , C(0), C(0)R 12 ,
C(0)(OR 13 ), P0 2 M,P(0)(OR 14 ), P(0)(R"), SO, S0 2 , C(0)(SR 13 ), or SSR 4 ; and
Y is CN, N 2 H 2 R 13 , N 2 HR 13 R 14 , N 3 , SCN, SCN 2 H 2 (R 15 ) 2 ,SC(0)N(R 15 ) 2 , SC(0)NHR 15 ,
S0 3 M, SR 4 , S0 2 M, P0 3 HM, P0 3 M 2 , P(0)(OR 15 )(OR 16 ), P(0)(OR l6 )(OM), P(0)(R I5 )(OR*),
P(0)(OM)R 15 , C0 2 M, C0 2 H, C0 2 R n , C(0)R 12 , C(0)(SR 13 ), SR 5 , or SSR 5 , or does not exist.
In yet further embodiments, R 5 , R 6 , R^R 9 , R 12 , R 13 , R M , R 15 , R 16 are the same or
different and are alkyls containing 1-12 carbon atoms; or C, or C2 connections to R 1 or R 3 in
cyclic derivatives;
X is CH 2 , 0, NH, NMe, S, S0 3 M, SH, SR 7 , S0 2 M, S(0)R 8 , S(0) 2 R 9 , S(0)OR 8 ,
S(0) 2 OR 9 , P0 3 M 2 , P(0)(OR 15 )(OR 16 ), P(0)(OR 16 )(OM), ?{0)^(OK% P0 3 HM or
P(0)(OM)R 15 ; and
Y is S0 2 M, S0 3 M, P0 3 HM, P0 3 M 2 , P(0)(OR 15 )(OR 16 ), P(0)(OR 16 )(OM), SR 5 , SR 4 or
SSR 5 , or does not exist.
In preferred embodiments, therapeutic compounds of the invention act as analgesic,
sedative and/ or anti-inflammatory agents. Preferred therapeutic compounds for use in the
invention include compounds having the formula (Formula II):
(Formula II)
R 3 C R 4
R
17_
■C R
18
R z C
R
-ON0 2
m
in which: m and n and p are integers from 0 to 10;
R 3 ' 17 are each independently hydrogen; a nitrate group; or A;
R 1,4 are each independently hydrogen; or A;
tare A is selected from: a substituted or unsubstituted aliphatic group (preferably a
branched, or sbai^ht-chain aliphatic moiety having from 1 to 24 carbon atoms in the chain,
which optionally may contain O, S, NR 6 and unsaturations in the chain, optionally bearing
from 1 to 4 hydroxy, or nitrate^ amino or aryl, or heterocyclic groups; an unsubstituted or
substituted cyclic aliphatic moiety haVing from 3 to 7 carbon atoms in the aliphatic ring,
which optionally may contain O, S, NR^anikunsaturations in the ring, optionally bearing
from 1 to 4 hydroxy, or nitrate, or amino or aryl, or heterocyclic groups; an unsubstituted or
substituted aliphatic moiety constituting a linkage offrom^O to 5 carbons, between R 1 and R 3
and/ or between R 17 and R 4 , which optionally may contain O, S^NR 6 and unsaturations in the
linkage, and optionally bearing from 1 to 4 hydroxy, or nitrate, or abaino or aryl, or
heterocyclic groups); a substituted or unsubstituted aliphatic group (preferably a branched,
9
cyclic or straigfifechain aliphatic moiety having from 1 to 24 carbon atoms in the chain),
J^^^ containing carbonyrh^kages (e.g., C = 0, C=S, C=NOH), which optionally may contain O,
S, NR 6 and unsaturations lisjhe chain, optionally bearing from 1 to 4 hydroxy, or nitrate, or
^£ . amino or aryl, or heterocyclic groups; a substituted or unsubstituted aryl group; a heterocyclic
5 group; amino (including alkylaminoxdialkylamino (including cyclic amino, diamino and triamino
moieties), arylamino, diarylamino, and al^ylarylamino); hydroxy; alkoxy; a substituted or
unsubstituted aryloxy.
R 2 , R\ R 18 , R 19 are optionally hydrogen; or A; or X-Y.
where X is F, Br, CI, N0 2) CH 2 , CF 2 , O, NH, NMe, CN, NHOH, N 2 H 3 , N 2 H 2 R 13 ,
10 N 2 HR 13 R 14 , N„ S, SCN, SCN 2 H 2 (R 15 ) 2 , SCN 2 H 3 (R 15 ), SC(0)N(R ,5 ) 2 , SC(0)NHR 15 , S0 3 M,
□ SH, SR 7 , S0 2 M, S(0)R 8 , S(0) 2 R 9 , S(0)OR 8 , S(0) 2 OR 9 , P0 2 HM, P0 3 HM, P0 3 M 2 ,
1 P(0)(OR 15 )(OR 16 ), P(0)(OR 16 )(OM), ?(0)(B^(OK\ P(0)(OM)R 15 , C0 2 M, C0 2 H, C0 2 R u ,
i] C(0), C(0)R 12 , C(0)(OR' 3 ), P0 2 H, P0 2 M, P(0)(OR 14 ), P(0)(R u ), SO, S0 2 , C(0)(SR 13 ), SR 5 ,
^ SSR 7 or SSR 5 ,
Wl5 Y is F, Br, CI, CH 3 , CF 2 H, CF 3 , OH, NH 2 , NHR 6 , NR 6 R 7 , CN, NHOH, N 2 H 3 ,
N 2 H 2 R 13 , N 2 HR 13 R 14 , N 3 , S, SCN, SCN 2 H 2 (R 15 ) 2 , SCN 2 H 3 (R 15 ), SC(0)N(R 15 ) 2 , SC(0)NHR 15 ,
S0 3 M, SH, SR 7 , S0 2 M, S(0)R 8 , S(0) 2 R 9 , S(0)OR 8 , S(0) 2 OR 9 , P0 2 HM, P0 3 M 2 ,
P(0)(OR 15 )(OR 16 ), P(0)(OR t6 )(OM), Pp^pR 8 ), P(0)(OM)R 15 , C0 2 M, C0 2 H, C0 2 R n ,
C(0)R 12 , C(0)(OR 13 ), C(0)(SR 13 ), SR 5 , SSR 7 or SSR 5 , or does not exist;
^Jfe^O R 6 , R 7 > R 8 "^R^ R 11 , R 12 , R 13 , R M , R 15 , R 16 are the same or different alkyl or acyl
0^*/^ groups containing 1-24 carb&qatoms which may contain 1-4 ON0 2 substituents; or Q - C 6
connections to R 1 - R 4 in cyclic derivatives; or are each independently hydrogen; a nitrate
group; or W.
M is H, Na + , K + , NH 4 + , N + H k R" (4 . k) where k is 0-3, or other pharmaceutical^
25 acceptable counterion;
and with the proviso that, when m = n = p = 1; R 19 , R 2 , R 18 , R 1 = H; R 17 , R 3 are nitrate
groups; that R 4 is not H or Q - C 3 alkyl.
In certain preferred embodiments, therapeutic compounds of the invention are
analgesic, sedative and/ or anti-inflammatory agents. Preferred therapeutic compounds for use
ill
"i
(
10
in the invention include compounds in which R 19 is X-Y. In a particularly preferred
embodiment: R 19 is X-Y and R\ R 6 , R 8 R 9 , R 10 , R 12 , R 13 , R 14 , R 15 , R 16 are the same or different
alkyl groups containing 1-24 carbon atoms which may contain 1-4 ON0 2 substituents, or C x
or d connections to R 1 - R 3 in cyclic derivatives; R 1 and R 3 are the same or different and
5 selected from H, Q-Q alkyl chains, which may inlude one O, linking R 1 and R 3 to form
pentosyl, hexosyl, cyclopentyl, or cycohexyl rings, which rings may optionally bear hydroxyl
substituents; R 2 and R 4 , are the same or different and selected from H, a nitrate group, C r C 4
alkyl optionally bearing 1-3 nitrate groups, and acyl groups (-C(O)R 5 ); R 7 , R 11 are the same or
different Cj- C 8 , alkyl or acyl..
10 In certain embodiments in which R 19 is X-Y, m, p = 1, and n = 0. In other
□ embodiments in which R 19 is X-Y, X is selected from CH 2 , O, NH, NMe, CN, NHOH,
| N 2 H 3 , N 2 H 2 R 13 , N 2 HR 13 R 14 , N 3 , S, SCN, SCN 2 H 2 (R 15 ) 2 , SCN 2 H 3 (R 15 ), SC(0)N(R 15 ) 2 ,
J SC(0)NHR 15 , S0 3 M, SH, SR 7 , S0 2 M, S(0)R 8 , S(0) 2 R 9 , S(0)OR 8 , S(0) 2 OR 9 , P0 3 HM,
^ P0 3 M 2 , P(0)(OR 15 )(OR 16 ), P(0)(OR 16 )(OM), Pp^XOR 8 ), P(0)(OM)R 15 , C0 2 M, C0 2 H,
! Jl5 C0 2 R u , C(0), C(0)R 12 , C(0)(OR 13 ), P0 2 M, P(0)(OR 14 ), P(0)(R 13 ), SO, S0 2 , C(0)(SR 13 ),
is* SSR 4 . In another embodiment in which R 19 is X-Y, Y is selected from CN, N 2 H 2 R 13 ,
|| N 2 HR 13 R 14 , N 3 , SCN, SCN 2 H 2 (R 15 ) 2 , SC(0)N(R 15 ) 2 , SC(0)NHR 15 , S0 3 M, SR 4 , S0 2 M,
I P0 3 HM, P0 3 M 2 , P(0)(OR 15 )(OR 16 ), P(0)(OR 16 )(OM), P(0)(R 15 )(OR 8 ), P(0)(OM)R 15 , C0 2 M,
3 C0 2 H, C0 2 R u , C(0)R 12 , C(0)(SR 13 ), SR 5 , SSR 5 , or does not exist. In a further
20 embodiment, X and/ or Y contain a sulfur-containing functional group. In certain preferred
embodiments, the compound of the invention comprises a heterocyclic functionality, more
preferably, a nucleoside or nucleobase. In further preferred embodiments, the compound of
the invention comprises a carbocyclic functionality, more preferably, a steroidal or
carbohydrate moiety.
11
In another aspect, a therapeutic compound of the invention is represented by the
formula (Formula III):
Y
X
R 3 C R 4
R
17
-C R
18
R' C-
I
■ONOo
m
in which: m is 1-10; R 1 ' 18 , X, and Y have the meaning as defined above. In certain preferred
embodiments, R 6 - R 16 are the same or different alkyl or acyl groups containing 1-24 carbon
atoms which may contain 1-4 ON0 2 substituents, or C x - C6 connections to R 1 - R 4 in cyclic
derivatives. In certain preferred embodiments, R 18 is A and n = 1.
In preferred embodiments, a therapeutic compound of the invention is represented by
the formula (Formula IV):
R 3 C R 4
R 17 C R
R 2 C ON0 2
R 1
in which: R 3 , R 1 =H; n, R^ 4 " 18 , X, and Y have the meaning as defined above. In
preferred embodiments, X is CH 2 or does not exist, and Y is selected from, F, Br, CI, CH 3 ,
CF 2 H, CF 3 , OH, NH 2 , NHR 6 , NR 6 R 7 , CN, NHOH, N 2 H 3 , N 2 H 2 R 13 , N 2 HR 13 R 14 , N 3 , S,
s18
SCN, SCNjHjCRJaSCNjHjfos), SC(0)N(R 15 ) 2 , SC(0)NHR 15 , S0 3 M, SH, SR 7) S0 2 M,
S(0)R 8) S{0) 2 K S(0)OR 8) S(0) 2 OR,, P0 2 HM, P0 3 M 2 , P(0)(OR 15 )(OR 16 ), P(0)(OR 16 )(OM),
P(0)(R 15 )(OR 8 ), P(0)(OM)R 15) C0 2 M, C0 2 H } C0 2 R U) C(0)R 12 , C(0)(OR 13 ), C(0)(SR 13 ),
SR 5 , SSR 7 or SSR 5 . In certain preferred embodiments, R 2 and R 4 are optionally H, a nitrate
group or a connection to R 5 -R 16 in cyclic derivatives.
In certain preferred embodiments, a compound of the invention is represented by the
formula (Formula V):
SSR 5
R 3 C—
-R 4
R 17 C-
-R 18
R 2 C—
-ON0 2
I,
m
n
in which m, n, R 1 " 18 , X, and Y have the meaning as defined above.
In another aspect, the invention includes novel compounds useful for treating pain,
mitigating inflammation, effecting analgesia and/or providing sedation. The compounds of
the invention can be represented by the structures shown hereinbelow, for example, the
structures of Formula III, IV and V. Novel compounds of the invention include nitrates Illr -
Illaj, IVn - Ivt, and Vd - Vag, whose syntheses are described in the following examples.
The invention also provides methods for treating a disease state associated with
inflammation, comprising administering to a subject an effective amount of a therapeutic
compound having a formula set forth above, such that a disease state associated with
inflammation is treated.
The invention further provides methods for treating a disease state or disorder in which
a level of sedation is desired, comprising administering to a subject an effective amount of a
sedative therapeutic compound having a formula set forth above, such that a disease state or
disorder is treated.
13
The invention provides methods for effecting analgesia comprising administering to a
subject an effective amount of a therapeutic compound having a formula set forth above, such
that analgesia is effected.
The invention further provides novel pharmaceutical compositions for treating pain,
mitigating inflammation, effecting analgesia and/or effecting sedation. A said pharmaceutical
composition comprises a therapeutic compound of the invention in an effective amount for
the particular indication and a pharmaceutical^ acceptable vehicle.
The invention also provides packaged pharmaceutical compositions for treating pain,
mitigating inflammation, effecting analgesia and/or effecting sedation. The packaged
pharmaceutical compositions include a therapeutic compound of the invention and
instructions for using the pharmaceutical composition for treatment of inflammation and/ or
pain.
BRIEF DESCRIPTION OF THE DRAWINGS
Figure 1 is a graph showing the effect of IVd neat (diamonds); with added L-cysteine
(2mM, triangles); with added dithiothreitol (2mM, DTT, squares); on soluble GCase activity
in rat aorta homogenate normalized to the maximal GTN response. Bars represent the mean
± standard errors calculated separately for each point.
Figure - ? is a graph showing the comparison of GTN (squares), Illm (circles) and IVh
(triangles) with added L-cysteine (1 mM) on soluble GCase activity in rat aorta homogenate
(a), and rat hippocampus homogenate (b). Data points represent the mean of duplicate
determinations carried out in identical GCase preparations.
Ftgttre-3-is a graph showing the comparison of GTN (squares), Va (circles) and
Vb (triangles) with added L-cysteine(l mM) on soluble GCase activity in rat aorta homogenate
homogenate (a), and rat hippocampus homogenate (b). Data points represent the mean ±
standard errors calculated separately for each point (n=8-ll).
■ Frgurc -4 is a graph showing the comparison of cyclic GMP accumulation in isolated rat
aorta induced by diluent (basal, open bar), GTN (filled bar), Va (stippled bar), or Illm
(hatched bar). Segments of rat aorta were exposed to diluent, 1 (aM drug (a), or 10|iM drug (b)
14
for 1 min and cyclic GMP content determined by radioimmunoassay. Data are the mean ±
standard errors ja, n=8; b, n=5).
is a graph showing the comparison of cyclic GMP accumulation in isolated rat
aorta induced by diluent (basal, open bar), GTN (filled bar), IVk (stippled bar ),Vb (cross-
hatched bar), or Vc (hatched bar). Segments of rat aorta were exposed to diluent, 1 \M drug
(a) , or 10|liM drug (b) for 1 min and cyclic GMP content determined by radioimmunoassay.
Data are the mean ± standard errors (a, n=5; b, n=4).
Fi g ur e 4 is a graph showing cyclic GMP accumulation in rat hippocampal slices
induced by diluent (basal, open bar), GTN (filled bar), and Va (stippled bar). Sections of rat
hippocampus (400 |im) were prepared and exposed to diluent, 10 [M drug (a) or 100 \xM drug
(b) for 3 min and cyclic GMP content determined by radioimmunoassay. Data are the mean ±
standard errors (a, n=4; b, n = 5).
Figure 7 is a graph showing the comparison of relaxation of isolated rat aorta induced
by GTN (squares), Va (open triangles), compound IVc (diamonds), compound IVd (open
squares), compound IVf (triangles), and compound IVg (open diamonds). Data points
represent the mean ± standard errors (n=5-8).
Figure 8 is a graph showing the comparison of relaxation of isolated rat aorta induced
by GTN (squares), IVk (open triangles), Vb (diamonds), Him (open squares), Vc (triangles),
and IVh (open diamonds). Data points represent the mean ± standard errors (n~3-8).
Figure 9 is a graph showing the comparison of the percent change in mean arterial
pressure (MAP) in conscious unrestrained rats after subcutaneous administration of 400
|amol/kg GTN (squares) or Va (open circles). Data points represent the mean ± standard
errors (n=6).
Figure 10 is a graph showing the comparison of the percent change in mean arterial
pressure in Inactin anaesthetized rats after intravenous bolus injection of GTN (squares) or Va
(open circles). Data points represent the mean ± standard errors (n=4).
Figure 11 is a graph showing the plasma levels (jxM) of Vb (circles) and its mononitrate
metabolite Vc (open squares) after subcutaneous administration of 200 |imol/kg Vb in
conscious unrestrained rats. Data points represent the mean of two experiments.
*
Figtmrt2"is a graph showing the relaxation induced by compound IVd (a) and IVc (b)
in untreated (squares) and GTN-tolerant (circles) isolated rat aorta. Aortae were made
tolerant by treatment with 0.5 mM GTN for 30 min. Data points represent the mean ±
standard deviation (n= 3-6).
Figwe-B is a graph showing the effect of Vm and Va in the mouse writhing test. GT
Vm produced a dose-dependent analgesic effect (A). Va also produced analgesia in the mouse
writhing test (B). Data are mean ± standard errors (n = 10-20).
Ei gu r e .- 14 -*s a graph showing the effect of Vm on paw flinches in rats after injection of
formalin into the footpad. In comparison to vehicle-treated control animals, Vm decreased
the initial pain response to formalin injection (at time = 0, *, p < 0.05), and the secondary
hyperalgia that developed between 20 and 40 minutes after formalin injection (A). For each
animal, a cumulative score (total number of flinches over 60 minutes) was calculated (B). Vm
significantly decreased cumulative paw flinches for 60 minutes after formalin injection. Data
are mean ± standard errors (N « 6-7).
Figb*e45 is a graph showing (A) the effects of chlormethiazole (CHLOR) and IVk
(200 )iM of each) on the membrane current induced by 10 |iM GAB A in a Xenopus oocyte
expressing the alply2L isoform of human recombinant GABA A receptors, and (B) the effect
of IVk on loss of the righting reflex in mice after intraperitoneal injection of 100 mg/kg and
200 mg/kg. Data in part B are mean ± standard error for three animals at each dose.
DETAILED DESCRIPTION OF THE INVENTION
In accordance with the invention there are provided methods and compositions useful
in the treatment of pain. Methods of the invention involve administering to a subject an
effective amount of a therapeutic compound which provides analgesia, mitigates inflammation
and/ or provides sedation. In some embodiments, the invention provides prophylactic
methods for avoiding or pre-empting pain, inflammation and the like in a subject. For
example, the subject may be susceptible or predisposed to these conditions, e.g., arthritic.
Alternatively, the subject may be undergoing a course of treatment, e.g. cancer chemotherapy,
16
which produces pain, inflammation or the like as a side effect. Methods of the invention may
be practiced prior to, concurrently with, or after such a course of treatment.
In accordance with the invention, analgesic, anti-inflammatory and/or sedative activity
can be effected by modulating an interaction with guanylyl cyclase (GCase; the enzyme
5 responsible for cGMP production in various areas of the body), and/ or by modulating levels
of cGMP and cAMP messenger molecules.
As used herein, the term "treating" pain encompasses preventing, ameliorating,
mitigating and/ or managing pain and/ or conditions that may cause pain, such as
inflammation. As used herein, "inhibiting" pain or inflammation encompasses preventing,
10 reducing and halting progression of same. The terms "organic nitrate" and "nitrate ester" are
□ used interchangeably herein, with no distinction drawn between them.
,p According to one aspect of the invention, there is provided a method for treating pain in
: J a subject, comprising administering to the subject an effective amount of a compound (nitrate
. jj ester) which effects analgesia in the subject. Preferably, analgesia is effected by stimulating
W15 GCase. In vivo, GCase activation is effected by nitric oxide (NO), the proximal activator of
1^ GCase, which is generated endogenously by enzyme action on arginine in response to many
; j5 biological triggers (}. R. Stone and M. A. Marietta, Biochemistry (1996) 35, 1093). One of the
^ major targets for organic nitrates is GCase activation, resulting in the production of cGMP.
;jj In this respect, organic nitrates act as NO-surrogates. In some cases, there is evidence that
20 organic nitrates may act also as NO-donors, but these two properties should be differentiated
and can be modulated by choice of the appropriate organic nitrate.
Experimental evidence obtained in a number of in vivo model systems supports the
notion that elevated levels of cGMP help effect analgesia. Sodium nitroprusside (SNP), which
releases NO non-enzymatically, blocked the hyperalgesic effect of prostaglandin (PGE^ in a
25 rat paw pressure test (Ferreira et al. 9 1991). Moreover, this effect was potentiated by an
inhibitor of cGMP phosphodiesterase, and blocked by an inhibitor of GCase (Ferreira et aL,
1991). The peripheral analgesic effects of morphine were attributed to elevations of cGMP
levels in sensory nerve fibres (Ferreira etaL, 1991; Granados-Soto etal. } 1997) in both the rat
paw pressure and formalin tests, since inhibition of GCase activity attenuated the analgesic
i
V
17
effects of locally applied morphine. Activation of the NO-cGMP system by NO donors such
as SNP has also been reported to potentiate beta-endorphin-induced analgesia in thermal tail-
flick test in mice (Xu etaL, 1995), an effect that was potentiated by a selective inhibitor
(zaprinast) of a cGMP-specific phosphodiesterase.
5 Sensitization of sensory nerve fibres leading to hyperalgesia is assumed to involve
increased concentrations of cAMP and calcium ions in sensory neurons, a process that may be
attenuated or counteracted by activation of the NO-cGMP pathway (Ferreira, 1993; Cunha et
al, 1999).
In accordance with another embodiment of the invention, administration of an effective
10 amount of a therapeutic compound to a subject effects analgesia in the subject by modulating
\ levels of cAMP and/ or cGMP. For example, it has been shown that NO-donors modulate
hyperalgesia via modulation of levels of cAMP, separately from and in addition to modulation
; of cGMP levels, in rat models of pain and nociceptor sensitization (Aley et al. 1998). Thus,
modulation of cAMP/cGMP levels is expected to be effective in inducing analgesia and in pain
15 management in individuals suffering injury, disease or aging.
In a further embodiment of the invention, there is provided a method for treating or
inhibiting inflammation in a subject, comprising administering to the subject an effective
amount of a compound which mitigates inflammation in the subject. Preferably,
inflammation is mitigated by modulation of levels of cGMP/cAMP. Inflammatory
20 hyperalgesia has been shown to be linked directly to the NO-cGMP pathway (Ferreira, 1993).
We have shown in our co-pending application USSN 09/267,379, filed March 15, 1999, vS ,
P which is hereby incorporated by reference, that novel nitrate esters have differential effects to ^ yo i c '*'
're-
activate soluble GCase and to cause cGMP accumulation in vascular and brain tissue. Further,
we have shown that the structure of the organic nitrate can be varied to alter potency and
25 efficacy towards both activation of GCase and accumulation of cGMP and effects resulting
from these processes in intact tissue, such as aortic strip relaxation. Activation of GCase and
accumulation of cGMP have been shown to be important in the induction of analgesia. We
show herein that novel organic nitrates are effective analgesics in animal models of pain
management. The mouse writhing model, with a relatively short time course of minutes, is a
18
preclinical model of acute pain; whereas formalin injection in the rat paw is a preclinical
model of acute and sensitization pain with a time course of minutes to hours, which
effectively mimics the hyperalgesia/allodynia underlying pain due to tissue damage (Yaksh,
1999).
5 In a further embodiment, the invention relates to a method for providing sedation
and/or anaesthesia in a subject, comprising administering to the subject an effective amount of
a compound (organic nitrate) which effects sedation or anaesthesia in the subject. In certain
aspects, the invention provides methods and compositions useful for reducing anxiety, and/or
aiding or inducing sleep.
10 y-Aminobutyric acid (GAB A) is the major inhibitory neurotransmitter in the
^ mammalian (including human) central nervous system. GAB A acts on three major classes of
:p neurotransmitter receptor, designated type A (GABAJ, type B (GABA B ) and type C
i jj (GAB A^. GABA A receptors play an important role in regulating many behavioural and
i J physiological functions. Thus, drugs that modulate GABA A receptor function are among the
j ^15 most widely used in clinical medicine. For example, drugs that selectively potentiate GABA A
\^ receptor function (such as the benzodiazepines) are extensively used to relieve anxiety,
;(l produce sedation and induce sleep. Given the importance of this receptor mechanism in
]% clinical medicine, there is a constant search for new chemical entities that modulate GABA A
^ function, as the currently available drugs have several side effects, including ataxia, amnesia,
20 tolerance and physical dependence. We show herein that organic nitrates that act as positive
allosteric modulators of GAB A A receptor function have sedative properties in the whole
animal that are comparable to known drugs. This effect of organic nitrates has not previously
been recognized or reported. Our findings provide direct evidence that nitrate esters are
useful as sedative agents. Such agents are useful as therapeutics for treating conditions such as,
25 for example, anxiety and pain associated with disease states; and as hypnotic agents.
According to the invention, nitrate esters may also be employed prophylactically, to prevent
or reduce anxiety, or to aid sleep.
Therapeutic compounds of the invention comprise at least one nitrate group. The
nitrate groups(s) can optionally be covalently bound to a carrier (e.g., an aromatic group, an
19
aliphatic group, peptide, steroid, nucleobase, nucleoside, peptidomimetic, steroidomimetic, or
nucleoside analogue, or the like). In addition to functioning as a carrier for the nitrate
functionality, the carrier molecule can enable the compound to traverse biological membranes
and to be biodistributed preferentially, without excessive or premature metabolism. Further,
in addition to functioning as a carrier for the nitrate functionality, the carrier molecule can
enable the compound to exert amplified analgesic, sedative, or anti-inflammatory effects
through synergism with the nitrate functionality.
In one embodiment, the invention provides a method comprising administering to a
subject an effective amount of a therapeutic compound which has at least one nitrate group
and is capable of effecting analgesia. In another embodiment, the therapeutic compound is
capable of mitigating inflammation. In a further embodiment, the therapeutic compound is
capable of effecting sedation. In the respective embodiments, the therapeutic compound has
the formula (Formula I):
wherein: E, Fj, F 2 , G^S^^re the same or different organic radicals which may be
joined in cyclic ring systems, and whicli s fi^y contain inorganic counterions.
In further aspects of the invention, therapeutic compounds of the invention effect
analgesia, effect sedation and/or mitigate inflammation in a subject to which the therapeutic
compound is administered, and have the formula (Formula II).
20
5 19
10
m5
20
25
R
,17_
,18
n
R C R'
I
-C R
-C— hON0 2
m
in which: m, n, p are integers from 0 to 10; R 3 * 17 are each independently hydrogen; a
nitrate group; or A; R 1 ' 4 are each independently hydrogen; or A; where A is selected from: a
substituted or unsubstituted aliphatic group (preferably a branched, or straight-chain aliphatic
moiety having from 1 to 24 carbon atoms in the chain, which optionally may contain O, S,
NR 6 andunsaturations in the chain, optionally bearing from 1 to 4 hydroxy, or nitrate, or
amino or aryl, or heterocyclic groups; an unsubstituted or substituted cyclic aliphatic moiety
having from 3 to 7 carbon atoms in the aliphatic ring, which optionally may contain O, S,
NR 6 and unsaturations in the ring, optionally bearing from 1 to 4 hydroxy, or nitrate, or
amino or aryl, or heterocyclic groups; an unsubstituted or substituted aliphatic moiety
constituting a linkage of from 0 to 5 carbons, between R 1 and R 3 and/or between R 17 and R 4 ,
which optionally may contain O, S, NR 6 and unsaturations in the linkage, and optionally
bearing from 1 to 4 hydroxy, or nitrate, or amino or aryl, or heterocyclic groups); a
substituted or unsubstituted aliphatic group (preferably a branched, cyclic or straight-chain
aliphatic moiety having from 1 to 24 carbon atoms in the chain), containing carbonyl linkages
(e.g., C = 0, C=S, C=NOH), which optionally may contain O, S, NR 6 and unsaturations in
the chain, optionally bearing from 1 to 4 hydroxy, or nitrate, or amino or aryl, or
heterocyclic groups; a substituted or unsubstituted aryl group; a heterocyclic group; amino
(including alkylamino, dialkylamino (including cyclic amino, diamino and triamino moieties),
arylamino, diarylamino, and alkylarylamino); hydroxy; alkoxy; a substituted or unsubstituted
aryloxy ; R 2 , R 5 , R 18 , R 19 are optionally hydrogen; or A; or X-Y; where X is F, Br, CI, N0 2 ,
CH 2 , CF 2 , O, NH, NMe, CN, NHOH, N 2 H 3 , N 2 H 2 R 13 , N 2 HR 13 R 14 , N 3 , S, SCN,
21
SCN 2 H 2 (R 15 ) 2 ,SCN 2 H 3 (R 15 ), SC(0)N(R 15 ) 2 , SC(0)NHR 15 , S0 3 M, SH, SR 7 , S0 2 M, S(0)R 8 ,
S(0) 2 R 9 , S(0)OR 8 , S(0) 2 OR 9 , P0 2 HM, P0 3 HM, P0 3 M 2 , P(0)(OR ,5 )(OR 16 ),
P(0)(OR 16 )(OM), P(0)(R IS )(OR , ) > P(0)(OM)R 15 , C0 2 M, C0 2 H, C0 2 R n , C(0), C(0)R 12 ,
C(0)(OR 13 ), P0 2 H, P0 2 M,P(0)(OR 14 ), P(0)(R 13 ), SO, S0 2 , C(0)(SR 13 ), SR 5 , SSR 7 or SSR 5 ;
5 Y is F, Br, CI, CH 3 , CF 2 H, CF 3 , OH, NH 2 , NHR\ NR 6 R 7 , CN, NHOH, N 2 H 3 , N 2 H 2 R 13 ,
N 2 HR 13 R 14 , N„ S, SCN, SCN 2 H 2 (R 15 ) 2 , SCN 2 H 3 (R 15 ), SC(0)N(R 15 ) 2 , SC(0)NHR 15 , S0 3 M,
SH, SR 7 , S0 2 M, S(0)R 8 , S(0) 2 R 9 , S(0)OR 8 , S(0) 2 OR 9 , P0 2 HM, P0 3 M 2 , P(0)(0R ,5 )(0R 16 ),
P(0)(OR 16 )(OM), ?(0)(K 15 )(OK\ P(0)(OM)R 15 , C0 2 M, C0 2 H, C0 2 R n , C(0)R 12 ,
C(0)(OR 13 ), C(0)(SR 13 ), SR 5 , SSR 7 or SSR 5 , or does not exist; R 6 , R 7 , R 8 R 9 , R 10 , R", R 12 , R 13 ,
10 R 14 , R 15 , R 16 are the same or different alkyl or acyl groups containing 1-24 carbon atoms which
[ft may contain 1-4 ON0 2 substituents; or C a - C 6 connections to R 1 - R 4 in cyclic derivatives; or
;fj are each independently hydrogen; a nitrate group; or A; M is H, Na + , K + , NH 4 + , N + H k R n (4 _ k)
'aI where k is 0-3, or other pharmaceutical^ acceptable counterion.
'si
;^ Preferred therapeutic compounds for use in the invention include compounds in which
;% R 19 is X-Y. In a particularly preferred embodiment: R 19 is X-Y and R 5 , R 6 , R 8 , R 9 , R 10 , R 12 , R 13 ,
|f R 14 , R 15 , R 16 are the same or different alkyl groups containing 1-24 carbon atoms which may
!U contain 1-4 ON0 2 substituents, or C { or C2 connections to R 1 - R 3 in cyclic derivatives; R 1
s g and R 3 are the same or different and selected from H, C { -C 4 alkyl chains, which may inlude
. &
*** one O, linking R 1 and R 3 to form pentosyl, hexosyl, cyclopentyl, or cycohexyl rings, which
20 rings may optionally bear hydroxyl substituents; R 2 and R 4 , are the same or different and
selected from H, a nitrate group, C r C 4 alkyl optionally bearing 1-3 nitrate group, and acyl
groups (-C(0)R 5 ); R 7 , R 11 are the same or different C x - C 8 , alkyl or acyl..
In certain embodiments in which R 19 is X-Y, m, p = 1, and n = 0. In other
embodiments in which R 19 is X-Y, X is selected from CH 2 , O, NH, NMe, CN, NHOH,
25 N 2 H 3 , N 2 H 2 R 13 , N 2 HR 13 R 14 , N 3 , S, SCN, SCN 2 H 2 (R 15 ) 2 , SCN 2 H 3 (R 15 ), SC(0)N(R 15 ) 2 ,
SC(0)NHR 15 , SO3M, SH, SR 7 , S0 2 M, S(0)R 8 , S(0) 2 R 9 , S(0)OR 8 , S(0) 2 OR 9 , P0 3 HM,
P0 3 M 2 , P(0)(OR 15 )(OR 16 ), P(0)(OR 16 )(OM), PpJ^pR 8 ), P(0)(OM)R 15 , C0 2 M, C0 2 H,
C0 2 R n , C(0), C(0)R 12 , C(0)(OR 13 ), P0 2 M,P(0)(OR 14 ), P(0)(R 13 ), SO, S0 2 , C(0)(SR 13 ),
SSR 4 . In another embodiment in which R 19 is X-Y, Y is selected from CN, N 2 H 2 R 13 ,
22
10
N 2 HR 13 R 14 , N 3 , SCN, SCN 2 H 2 (R 15 ) 2 , SC(0)N(R 15 ) 2 , SC(0)NHR 15 , S0 3 M, SR\ S0 2 M,
PO3HM, P0 3 M 2 , P(0)(OR 15 )(OR 16 ), P(0)(OR 16 )(OM), Ppj^XOR 8 ), P(0)(OM)R 15 , C0 2 M,
C0 2 H, C0 2 R n , C(0)R 12 , C(0)(SR 13 ), SR 5 , SSR 5 , or does not exist. In a further
embodiment, X and/or Y contain a sulfur-containing functional group. In certain preferred
embodiments, the compound of the invention comprises a heterocyclic functionality, more
preferably, a nucleoside or nucleobase. In further preferred embodiments, the compound of
the invention comprises a carbocyclic functionality, more preferably, a steroidal or
carbohydrate moiety.
In another aspect of the invention, a therapeutic compound of the invention is
represented by the formula (Formula III):
ill
us
20
25
R 3 C R 4
5 17_
-C R
18
R C
R
■ONO,
m
in which: m, n are 1-10; R 1 ' 18 , X, and Y have the meaning as defined above. In certain
preferred embodiments, R 6 - R 16 are the same or different alkyl or acyl groups containing 1-24
carbon atoms which may contain 1-4 ON0 2 substituents, or C, - C6 connections to R 1 - R 4 in
cyclic derivatives. In certain preferred embodiments, R 18 is A and m = n = 1. In further
preferred embodiments, therapeutic compounds of the invention have a formula selected from
(Formulae Ilia - Illam):
-F
Ha
-ON0 2
-ONO,
23
25
27
Illam
I>
ONO,
In a further aspect of the invention, a therapeutic compound of the invention is
represented by the formula (Formula IV): Y
■R 4
>17_
-R 18
-ON0 2
n
■C-
C-
R 1
in which n = 0, X is CH 2 or does not exist, and Y is selected from, F, Br, CI, CH 3 ,
CF 2 H, CF 3) OH, NH 2) NHR 6 , NR 6 R 7 ,CN, NHOH, N 2 H 3 , N 2 H 2 R 13 , N 2 HR 13 R 14 , N 3 , S,
SCN, SCN 2 H 2 (R 15 ) 2 ,SCN 2 H 3 (R 15 ), SC(0)N(R 15 ) 2 , SC(0)NHR 15 , S0 3 M, SH, SR 7 , S0 2 M,
S(0)R 8) S{0)&, S(0)OR 8 , S(0) 2 OR„ P0 2 HM, P0 3 M 2 , P(0)(OR 15 )(OR 16 ), P(0)(0RJ(0M),
PCO^XOR,), P(0)(OM)R 15 , C0 2 M, C0 2 H, C0 2 R n> C(0)R 12 , C(0)(OR 13 ), C(0)(SR 13 ),
SR 5 , SSR 7 or SSR 5 . R 2 , R 4 , R 5 , R 6 , R 7 , Rg^Rg, Rio) Rn> Ri2> Rn> Ru> Ris> an d Ri6 are as defined
above. In certain preferred embodiments, R 2 and R 4 are optionally H, a nitrate group or a
connection to R 5 -R 16 in cyclic derivatives.
In certain preferred embodiments, a compounds of the invention is represented by the
formula (Formulae IVa-IVt):
iva r~ cl
-ON0 2
-ONO,
30
10
'a*
IVb
IVc
IVd
-SCN
-ON0 2
-ON0 2
OCH 2 CH 3
-ON0 2
-ON0 2
OCH 2 CH 3
A&5
IVe
20
-S 2 0 3 Na
■ON0 2
J
25
IVg
IVh
J
0 3 NO
J
0 2 N0
IVi
CN
-0N0 2
■0N0 2
10
IVj
-Br
-ON0 2
-ON0 2
•4
i J
J15
it)
•
: -0
20
25
IVk
IVI
IVm
IVn
0,NO
\
II/ 0 --.
-ON0 2
-ON0 2
r
■S0 2 Ph
■ON0 2
-ON0 2
SCN
ONO,
•
33
In a further aspect of the invention, compounds according to the invention are
represented by the formula (Formula V):
SSR 5
R 3 C R 4
>17_
-C R
18
R' C
R
■ONOo
m
>ea in which R 2 is optionally H or a connection to R 5 in cyclic derivatives, R 4 is H or a
ir=J
! Q 5 nitrate group, and R 5 is as described above.
l ij In certain preferred embodiments, compounds of the invention are represented by the
formula (Formulae Va-Vag):
liilO Va
■s — s-
-ono 5
-ONOo
-ONOo
-ONOp
Vb
15
s — s
0 2 N0
0N0 3
20 Vc
s s
0 OH
N* O'
//
0
37
38
Vac
-S S-
■ONO,
-OH
-ONO,
-ONO,
10
'■■-J'
20
Vad
-S S
Vae
ijJl5 Vaf
Vag
3^2
In another aspect, the invention provides novel compounds which can be represented by
25 structures of Formula III, Formula IV, and Formula V. Table 1 lists data associated with these
compounds using art-recognized characterization techniques. Further, the invention provides
novel pharmaceutical compositions comprising a therapeutic compound (nitrate ester) of the
invention and a pharmaceutically acceptable vehicle.
39
Table 1
'HNMR
,3 C NMR
Ela
(CDC1,): 5.34-5.57 (1H, dm, 3 J HF 20.6), 4.53-4.87
(4H, superposition several multiplets, OjNO-CHj
+ CH 2 F, 2 J HF 46.7, 4 J HF 0.66)
(CDClj): 79.47 (d, 'J CF 177), 76.73 (d, J J CF
20.6), 67.84 (d, 3 J CF 6.87)
EIc
(CDC1,): 5 5.7 (1H, t, 2 J HF 54), 5.45 (1H, m), 4.5-4.9
(2H,m), 4.15-4.35 (1H, m)
(CDClj): 8 75.55, 68.05, 60.76
ma
(CDClj): 5 5.46 (1H, m), 4.804.87 (1H, dd, J 3.5,
12.9), 4.65-4.72 (1H, dd, J 6.2, 12.9), 3.7-3.8 (2H, m)
(CDClj): 8 77.24, 68.57, 39.86
mf
(CDClj) 5 8.72 (s, 1H), 5.38 (t, 1H), 4.6 (d, 2H),
2.45 (s, 3H)
mg
pMSOd 6 ) CHON0 2 only: 5 4.8-5.8
pMSOd 6 ) CONOj only: 8 85.68, 84.17,
82.47, 76.50
mh
(CD 3 OD) 8 4.85 (3H, m), 3.5 (1H, m)
(CDjOD) 8 70.61, 36.74
mi
(CDClj): 8 6.95 (dd, 1H), 6.71 (dd, 1H), 6.09 (m,
1H), 3.80 (dd, 1H), 3.32 (dd, 1H)
(CDClj): 8 137.9, 132.5, 76.6, 52.9
mj
(CDC1,): 5 5.62 (2H, m), 3.60 (4H, m)
(CDClj): 8 77.87, 25.22
mk
(CD,CN): 8 3.45 (m, 2H), 5.72 (m, 2H)
(CDjCN): 8 79.98, 28.87
mi
(CDjCN): 8 79.48, 33.45, 28.47
mm
(DMSOd 6 ): 8 5.97 (m, 2H), 3.80 (m, 4H)
(DMSOd 6 ): 8 78.84, 52.60
mn
(CDClj): 8 5.73 (m, 1H), 4.62 (m, 1H), 3.96-3.77
(m, 1H), 3.58-3.32 (m, 1H)
(CDClj): 8 81.47, 57.85, 53.50, 38.75
mo
(CDClj): 8 81.24, 69.79, 33.26, 27.24
mp
(CDClj): 8.5.36 (m, 1H), 3.11-3.60 (m, 4H), 2.33
(m,2H)
(CDClj): 8 78.92, 33.66, 30.64, 27.36
mq
(CDC1,): 8 5.47 (m, 1H), 3.53-3.05 (m, 4H), 2.29
(m,2H)
(CDClj): 8 81.32, 37.12, 32.97, 30.98
mw
31 P(CDC1 3 ,
162 MHz)
24.60
(CDClj, 300 MHz): 5.31-5.45 (m, 1H), 3.92-4.08 (m,
4H), 3.63-3.81 (m, 2H), 2.03-2.30 (m, 2H), 1.16-1.24
(superposition of 2 1, 6H, J 7)
(CDClj, 75 MHz): 76.83, 62.15 (d, J 6.37),
43.77 (d, J 8.95), 27.08 (d, J 142.00), 15.99
(d, J 5.88)
40
mx 31 p
(CD 3 OD
122 MHz)
17.62
(CDjOD 300 MHz): 5.38-5.63 (m, 1H), 3.75-4.25
(superposition of 2 m, 4H), 1.88-2.20 (m, 2H), 1.12-
1.28 (t, 3H)
(CDjOD 75 MHz): 81.14, 61.17 (d, J 5.41),
45.56 (d, J 5.94), 29.35 (d, J 131.74), 17.00
(d, J 6.75)
mak
'H (CD 3 CN 300 MHz): 7.8 (s, 1H), 5.75-5.85 (m,
1H), 4.90-4.97 (dd, 1H, J 12.87, 3.39), 4.54-4.76 (m,
3H), 3.46 (s, 3H), 3.27 (s, 3H)
13 C (CD 3 CN 75 MHz) (for polynitrated
chain): 78.51, 70.58, 46.01, 30.10, 28.18
Ivi
(CDC1 3 ): 8 5.45 (1H, m), 4.83 (1H, dd), 4.65 (1H,
dd), 2.9 (2H, m)
(CDjOD): 8 116.44, 75.37, 71.20, 19.19
Ivk
(CDC1 3 ) 5 8.55 (s, 1H), 4.55 (t, 2H), 3.15 (t, 2H),
2.37 (s, 3H)
(CDClj) 8 150.9, 150.7, 125.3, 72.53, 24.47,
15.18
Ivm
(CDClj): 5 7.5-8.0 (arom, 5H), 5.7 (1H, m), 4.94
(1H, dd), 4.62 (1H, dd), 3.5 (2H, m)
(CDClj): 8 135.45, 134.79, 129.81, 27.95,
73.08, 70.04, 54.73
Ivs
1H-NMR(CDC13, 300 MHz): 5.23-5.32 (1H, m),
4.87 (1H, dd, J 12.82, 3.22), 4.68 (1H, dd, J 12.83,
6.09), 2.77-2.94 (2H, m), 1.66 (1H, t, J 9.07)
13C-NMR: (CDC13, 75.48 MHz): 79.39,
69.30, 23.68
Ivt
1H-NMR(CDC13, 300 MHz): 5.29-5.38 (1H, m),
4.76 (1H, dd, J 12.94, 3.11), 4.55 (1H, dd, J 12.94,
6.37), 3.30 (1H, dd, J 14.06, 5.98), 3.13 (1H, dd, J
14.61, 6.35)
13C-NMR: (CDC13, 75.48 MHz): 194.10,
77.00, 69.79, 30.42, 27.78
Vb
(CDClj) 5 5.56 (m, 2H), 3.38-2.95 (m, 4H)
(CDjOD) 8 85.93, 32.77
Vc
(CDClj): 5 5.85-5.91 (1H, m), 4.50-4.58 (1H, m),
3.22-3.29 (1H, dd, J 5.47, 12.78), 2.97-3.05 (1H, dd, J
4.6, 11.88), 2.82-2.90 (1H, dd, J 2.87, 12.78), 2.74-
2.83 (1H, dd, J 3.15, 11.9)
(CDClj): 8 87.6, 74.96, 36.20, 31.54
Ve
(CDClj): 8 7.44-7.51 (m, arom 2H), 7.17-7.24 (d,
arom 2H, J 7.91), 5.47-5.59 (m, 1H), 4.83-4.93 (dd,
1H, J 12.81, 2.78), 4.57-4.67 (dd, 1H, J 12.82, 5.71),
3.02-3.12 (dd, 1H, J 14.48, 6.01), 2.9-2.99 (dd, 1H, J
(CDClj): 8 21.53, 36.78, 69.82, 77.68,
130.52, 130.62, 132.55, 139.23
i •
14.47, 7.72), 2.38 (s, 3H)
Vf
(CDC1,): 6 7.48-7.57 (m, arom 2H), 7.48-7.57 (m,
arom 2H), 5.49-5.59 (m, 1H), 4.84-4.93 (dd, 1H, J
12.79, 2.79), 4.58-4.68 (dd, 1H, J 12.79, 5.75), 3.84
(s, 3H), 3.02-3.12 (dd, 1H, J 14.47, 5.8), 2.89-2.99
(dd, 1H, J 14.46, 7.99).
(CDC1 3 ): 5 36.57, 55.87, 69.75, 77.76,
115.47, 126.71, 133.76, 160.94.
Vg
(CDClj): 8 7.47-7.54 (m, arom 2H), 7.32-7.38 (m,
arom 2H), 5.45-5.55 (m, 1H), 4.844.97 (dd, 1H, J
12.86, 2.92), 4.58-4.68 (dd, 1H, J 12.86, 5.68), 2.91-
3.11 (m, 2H).
(CDClj): 5 36.87, 69.80, 77.5, 129.98,
130.85, 134.51, 134.79.
Vh
(CDClj): 5 8.21-8.27 (m, arom 2H), 7.67-7.74 (m,
arom 2H), 5.44-5.54 (m, 1H), 4.864.94 (dd, 1H, J
12.92, 3.11), 4.61-4.70 (dd, 1H, J 12.92, 5.56), 3.01-
3.16 (m, 2H).
(CDCI3): 8 37.24, 69.75, 77.41, 124.82,
127.26, 144.83
Vi
(CDClj: 5 7.40-7.55 (m, arom 4H), 5.44-5.55 (m,
1H), 4.854.92 (dd, 1H, J 12.87, 2.91), 4.60-4.70 (dd,
1H, J 12.86, 5.66), 2.92-3.11 (m, 2H).
(CDCI3): 8 36.87, 69.79, 77.48, 122.72,
130.93, 132.90, 135.15.
Vj
(CDC1,): 5 7.55-7.62 (d, arom 2H, J 7.16), 7.29-7.44
(m, arom 3H), 5.46-5.58 (m, 1H), 4.82-4.92 (dd,
1H, J 12.85, 2.79), 4.574.67 (dd, 1H, J 12.86, 5.67),
3.01-3.13 (dd, 1HJ 14.51, 6.24), 2.92-3.02 (dd, 1H, J
14.52, 7.4)
(CDClj): 8 36.97, 69.88, 77.61, 128.60,
129.50, 129.85, 136.02
Vk
(CDC13, 300 MHz): 8.09 (1H, dd, J 8.12, 0.36), 8.02
(1H, dd, J 7.8, 1.15), 7.51-7.59 (1H, m, J 7.24, 1.44),
7.21-7.29 (1H, m, J 7.35, 0.54), 5.40-5.49 (1H, m),
(CDC13, 75.48 MHz): 166.21, 140.41,
132.78, 131.46, 127.63, 125.48, 77.25, 71.03,
61.42, 32.51,28.29, 14.17
4.70-4.78 (1H, ddj 13.04, 2.95), 4.33-4.45 (3H, m,
superposition of 1H from CH2-ON02 and quartet
from 0-CH2-CH3), 2.66-2.87 (2H, quartet, J 6.92),
1.39(3H,t,J7.14)
VI
(CDClj, 300MHz): 8 7.97 (s, 1H), 7.48-7.51 (d, 1H),
7.17-7.22 (m, 1H), 6.84-6.89 (d, 1H), 6.05 (s, 2H),
5.48-5.58(m, lH),4.81-4.89 (dd, 1H), 4.53-4.61 (dd,
1H), 4.25-4.4 (m, 2H), 3.05-3.10 (m, 2H), 1.35-1.45
(t,3H).
(CDC1 3 , 75.48 MHz): 8 14.17, 36.27, 62.15,
69.71, 76.57, 101.77, 108.27, 110.31, 125.20,
127.55, 127.87, 146.87, 147.811, 149.76,
165.89
Vm
(CDCl 3 ,300MHz): 5 8.04-8.11 (m, arom 2H), 7.55-
7.62 (m, arom 1H), 7.30-7.34 (m, arom 1H), 5.43-
5.54 (m, 1H), 4.88-4.97 ( dd, 1H, J 12.95, 2.79),
4.62-4.71 (dd, 1H, J 12.94, 5.35 ), 4.45-4.39 (q, 2H, J
7.12), 2.92-3.08 (m, 2H), 1.39-1.47(t, 3H, J 7.13)
(CDC1 3 ,75.48 MHz): 5 14.17, 26.23, 35.95,
61.55, 69.54, 77.24, 125.56, 125.92, 127.91,
131.52, 132.93, 139.56, 166.189
Vn
(CDC1„ 300MHz): 5 8.92-8.97 (m, arom 1H), 8.07-
8.23 (m, arom 2H), 7.46-7.77 (m, arom 3H), 5.53-
5.62 (m, 1H), 4.91-4.99 (dd, 1H, J 12.98, 2.77), 4.61-
4.7 (dd, 1H, J 12.98, 5.35), 3- 3.18 (m, 2H)
(CDC1„75.48 MHz): 8 36.06, 69.62, 70.24,
77.42, 121.97, 125.65, 126.43, 126.70,
128.52, 135.31, 136.37, 145.62, 149.65
Vo
(CDC1 3 , 300MHz): 5 7.55-7.59 (m, arom 1H), 7.40-
7.45 (m, arom 1H), 7.25-7.36 (m, arom 2H), 5.45-
5.55 (m, 1H), 4.854.95 (dd, 1H, J 12.98, 3), 4.59-
4.69 (dd, 1H, J 12.89, 6.67), 2.95-3.12 (m, 2H)
(CDC1 3 ,75.48 MHz): 8 37.10, 69.87, 77.44,
126.80, 128.38, 128.52, 130.83, 135.76,
138.01
Vp
(CDC1 3 , 300MHz): 5 7.65-7.67 (d, arom 1H J 2.15),
7.44- 7.47 (d, arom 1H, J 7.42), 7.28- 7.4 9 (m, arom
1H), 5.46-5.54 (m, 1H), 4.87- 4.94 (dd, 1H, J 12.89,
2.99), 4.61-4.68 (dd, 1H, J 12.88, 5.65), 2.95-3.12
(m,2H)
(CDC13 75.48 MHz): 8 36.59, 69.32, 76.90,
127.49, 129.86, 131.04, 132.31, 133.59,
135.65
Vq
(CDC1 3 , 300MHz): 5 7.2-7.6 (m, arom 8H), 5.42-
5.56 (m, 2H), 4.82-4.95 (dd, 2H), 4.55- 4.67 (dd,
2H), 2.93-3.15 (m,4H)
43
Vr
(CDC13, 300MHz): 5 8.63 (s, arom 1H), 8.01-8.13
(m, arom 2H), 7.57-7.65 (m arom 1H), 7.26- 7.37
(m, arom 1H), 5.42- 5.51 (m, arom 1H), 4.88-4.97
(dd, 1H, J 12.9, 2.7), 4.62-4.71 (dd, 2H, J 12.9, 5.1),
4.49- 4.57 (t, 2H, J 6.6), 3.23-3.32 (t, 2H, J 6.6),
2.91-3.08 (m, 2H), 2,45 (s, 3H)
Vs
(oVacetone), 300 MHz): 8.16-8.23 (1H, d, J 7.72),
8.07-8.13 (1H, dd, J 7.72, 1.11), 7.63-7.72 (1H, m, J
8.38, 1.26), 7.33-7.42 (1H, m, J 7.33), 5.67-5.76 (1H,
m), 5.09-5.17 (1H, dd, J 12.91, 2.68), 4.83-4.93 (1H,
dd, J 12.91, 5.91), 3.18-3.32 (2H, m)
(ds-acetone, 100.62 MHz): 167.74, 134.01,
132.64, 126.62, 126.21, 78.90, 71.69, 36.79
Vt
(CDC1 3 , 300MHz): 5 7.53-7.59 ( d, arom 1H, J
8.35), 7.46-7.49 (d, arom 1H, J 1.82), 7.37-7.43 (m,
arom 1H), 6.24 (s, 1H), 5.46-5.55 (m, 1H), 4.86-4.94
(dd, 1H, 12.92, 2.92), 4.61-4.69 (dd, 1H, J 12.93,
5.75), 3.0-3.15 (m, 2H), 2.42 (s, 3H)
(CDC1„ 75.48 MHz): 8 19.01, 37.10, 70.01,
77.46, 115.27, 115.34, 119.52, 123.07,
125.83, 141.13, 152.38, 154.25, 160.51
Vu
(CDC1 3 , 300MHz): 5 7.55-7.58 (m, arom 1H), 7.40-
7.45 (m, arom 1H), 7.27-7.32 (m, arom 2H), 5.46-
5.54 (m, 1H), 4.86-4.94 (dd, 1H, J 12.88, 2.92), 4.59-
4-68 (dd, 1H, J 12.88, 5.65), 2.95-3.13 (m, 2H)
(CDC13, 75.48 MHz): 5 37.08, 69.83, 77.40,
lZO.oU, 1/0.4U, 1zo.j4, IjU.oj, Ijj./o,
137.98
Vv
(CDCI3, 300 MHz): 8.09-8.14 (1H, d, J 8.15), 7.98-
8.04 (1H, dd, J 7.75, 0.91), 7.52-7.59 (1H, m, J 8.22,
I. 19), 7.22-7.28 (1H, m J 7.65), 4.53-4.61 (1H, dd, J
II. 33, 3.63), 4.42-4.50 (1H, m, J 11.33, 6.27), 4.32-
4.41 (2H, qu., J 7.13), 4.164.25 (1H, m), 2.74-2.84
(2H, m), 2.55-2.73 (1H, br.s.), 1.34-1.42 (3H, t, J
7.13)
(CDClj, 75.48 MHz): 166.34, 140.25,
132.90, 131.49, 127.67, 125.60, 74.34, 66.83,
61.53, 40.89, 14.20
Vx
(CDClj, 300MHz): 5 5.49-5.61 (m, 1H), 4.88-4.90
(m, 1H), 4.62-4.74 (m, lH),4.40-4.55 (m, 1H),3.59-
3.78 (m, superpos. 5H), 2.69-3.21 (m, superpos.
5H), 2.14-2.29 (m, 1H), 1.90-2.12 (m, 3H), 1.22-1.28
(CDClj, 75.48 MHz): 5 17.39, 25.20, 29.40,
36.41/36.51, 38.55, 41.94/42.19, 47.32,
52.56, 59.12, 69.90/70.29,77.89/78.00,
173.01, 173.48
44
(d,3H)
Vy
(CDC1„ 300 MHz): 5 8.5 (s, 1H), 2.94-3.02 (t,
ZnJ,Z.OZ-Z./Z (q, ZriJ, 1.33 (S, 3ri), 1.jo-1.4j (t,
1H).
(CDClj, 75.48 MHz): 8 15.41, 26.41, 31.51,
39.72, 129.36, 149.90
Vz
(CDjCN, 300 MHz): 4.60-4.68 (1H, dd, J .11.44,
3.36), 4.43-4.52 (1H, dd, J 11.35, 6.97), 4.114.21
(1H, m), 2.80-3.00 (2H, m)
(CD,CN, 75.48 MHz): 76.33, 67.34, 42.74
Vaa
(CDClj, 300MHz): 8 8.05-8.15 (m, arom 2H), 7.55-
7.65 (m, arom 1H), 7.25-7.35 (m, arom 1H), 5.40-
5.55 (m, 1H), 4.85-4.95 (dd, 1H), 4.60-4.70 (dd, 1H),
3.95 (s, 3H), 2.90-3.10 (m, 2H)
(CDClj, 5.48 MHz): 8 36.42, 52.87, 69.98,
77.69, 126.07, 126.45, 128.01, 132.06,
133.53, 140.161, 167.10
Vab
(CDC1 3 , 300MHz): 5 7.30-7.39 (m, arom 5H), 5.28-
5.37 (m, 1H), 4.72-4.78 (dd, 1H, J 12.86, 2.79), 4.45-
4.53 (dd, 1H, J 12.86, 5.9), 3.95 (s, 2H), 2.44-2.52
(dd, 1H, J 14.38, 6.1), 2.33-2.42 (dd, 1H, J 14.38,
7.34)
. (CDClj, 75.48 MHz): 8 32.01, 35.997,
43.66, 69.89, 77.72, 128.33, 129.22, 129.81,
137.22
Vac
1H-NMR(CDC13, 300 MHz): 5.48-5.58 (1H, m),
4.89 (1H, dddj 12.91, 2.91, 1.21), 4.56-4.70 (2H,
m), 4.48 (1H, dd, J 11.41, 6.4), 4.164.26 (1H, m),
2.94-3.12 (2H, m), 2.81-2.91 (2H, m)
13C-NMR: (CDC13, 75.48 MHz): 77.30,
74.22, 69.46, 69.38, 66.90, 66.78, 42.12,
41.99, 36.82, 36.59
Vad
(CDClj, 400 MHz): 5.55-5.65 (m, 1H), 4.874.94
(dd, 1H, J 12.94, 2.94), 4.624.70 (m, 1H, J 12.88),
3.13-3.30 (m, 2H)
(CDClj, 400 MHz): 76.74, 69.46/69.42,
36.65/36.63
It will be noted that the structure of some of the compounds of this invention include
asymmetric carbon atoms. It is to be understood accordingly that the isomers (e.g.,
5 enantiomers, diastereomers) arising from such asymmetry are included within the scope of
this invention. Such isomers can be obtained in substantially pure form by classical separation
techniques and by asymmetric synthesis (for example, see below in Example 21). Unless
45
expressly noted to the contrary, compounds referred to herein shall be construed to include
both the R and S stereoisomers at each stereogenic centre.
In certain embodiments, a therapeutic compound of the invention comprises a cation
(i.e., in certain embodiments, one of X or Y includes a cation, e.g., in the compound of
5 formula IVd). If the cationic group is a proton, then the compound is considered an acid. If
the proton is replaced by a metal ion or its equivalent, the compound is a salt.
Pharmaceutical^ acceptable salts of the therapeutic compound are within the scope of the
invention. For example, M can be a pharmaceutical^ acceptable alkali metal (e.g., Li, Na, K),
ammonium, alkaline earth metal (e.g., Ca, Ba, Mg), higher valency cation, or polycationic
10 counter ion (e.g., polyammonium cation) (see e.g., Berge et al. (1977)). It will be appreciated
that the stoichiometry of an anionic portion of the compound to a salt-forming cation will
vary depending on the charge of the anionic portion of the compound and the charge of the
counterion. Preferred pharmaceutical^ acceptable salts include a sodium, potassium, or
s l calcium salt, but other salts are also contemplated within their pharmaceutical^ acceptable
ijl5 range.
^ Therapeutic compounds of the invention can be administered in a pharmaceutical^
*j acceptable vehicle. As used herein "pharmaceutical^ acceptable vehicle" includes any and all
y
S solvents, excipients, dispersion media, coatings, antibacterial and antifungal agents, isotonic
Q and absorption delaying agents, and the like which are compatible with the activity of the
20 compound and are physiologically acceptable to the subject. An example of a
pharmaceutical^ acceptable vehicle is buffered normal saline (0.15 M NaCl). The use of such
media and agents for pharmaceutical^ active substances is well known in the art. Except
insofar as any conventional media or agent is incompatible with the therapeutic compound,
use thereof in the compositions suitable for pharmaceutical administration is contemplated.
25 Supplementary active compounds can also be incorporated into the compositions.
Carrier or substituent moieties useful in the present invention may also include moieties
which allow a therapeutic compound to be selectively delivered to a target organ. For
example, delivery of a therapeutic compound to the brain may be enhanced by a carrier
moiety using either active or passive transport (a "targeting moiety"). Illustratively, the
46
carrier molecule may be a redox moiety, as described in, for example, U.S. Patents 4,540,654
and 5,389,623, both to Bodor. These patents disclose drugs linked to dihydropyridine
moieties which can enter the brain, where they are oxidized to a charged pyridinium species
which is trapped in the brain. Thus drugs accumulate in the brain. Other carrier moieties
5 include compounds, such as amino acids or thyroxine, which can be passively or actively
transported in vivo. Such a carrier moiety can be metabolically removed in vivo> or can
remain intact as part of an active compound. Structural mimics of amino acids (and other
actively transported moieties) including peptidomimetics, are also useful in the invention. As
used herein, the term "peptidomimetic" is intended to include peptide analogs which serve as
10 appropriate substitutes for peptides in interactions with e.g., receptors and enzymes. The
peptidomimetic must possess not only affinity, but also efficacy and substrate function. That
is, a peptidomimetic exhibits functions of a peptide, without restriction of structure to amino
*i acid constituents. Peptidomimetics, methods for their preparation and use are described in
^ Morgan et ai, (1989) "Approaches to the discovery of non-peptide ligands for peptide
^115 receptors and peptidases". In Annual Reports in Medicinal Chemistry (Vinick, F.J., ed.) pp. 243-
^ 252, Academic Press, San Diego, CA. Many targeting moieties are known, and include, for
U
{J example, asialoglycoproteins (see e.g., Wu, U.S. Patent 5,166,320) and other ligands which are
=±
transported into cells via receptor-mediated endocytosis (see below for further examples of
targeting moieties which may be covalently or non-covalently bound to a target molecule).
20 In the methods of the invention, pain and/ or inflammation in a subject is mitigated by
administering an analgesic, sedative or anti-inflammatory therapeutic compound of the
invention to the subject. The term "subject" is intended to include living organisms in which
pain can occur. Examples of subjects include humans, apes, monkeys, cows, sheep, goats, dogs,
cats, mice, rats, and transgenic species thereof. Administration of the compositions of the
25 present invention to a subject to be treated can be carried out using known procedures, at
dosages and for periods of time effective to alleviate pain in the subject. An effective amount
of the therapeutic compound necessary to achieve a therapeutic effect may vary according to
factors such as the subject, the age, sex, and weight of the subject, and the ability of the
therapeutic compound to mitigate pain and inflammation in the subject. Dosage regimens can
be adjusted to provide the optimum therapeutic response. For example, several divided doses
may be administered daily or the dose may be proportionally reduced as indicated by the
exigencies of the therapeutic situation. A non-limiting example of an effective dose range for a
therapeutic compound of the invention (e.g., Va) is between 0.5 and 5000 mg/kg of body
weight/per day, preferably between 50 and 1000 mg/kg/day, and still more preferably
between 250 and 750 mg/kg/day. In an aqueous composition, preferred concentrations for
the active compound (i.e., the therapeutic compound that can mitigate pain) are between 5 and
500 mM, more preferably between 10 and 100 mM, and still more preferably between 20 and
50 mM.
According to the invention, therapeutic compounds are administered to a subject by a
route which is effective for mitigating inflammation, effecting analgesia and/ or effecting
sedation. Suitable routes of administration include but are not limited to sublingual, oral,
buccal, transdermal, nasal, subcutaneous, intraocular, intravenous, intramuscular and
intraperitoneal (e.g., by injection). Preferred routes of administration are oral and
transdermal. The therapeutic compounds can be administered with a pharmaceutically
acceptable vehicle. Depending on the route of administration, the active compound may be
coated in a material to protect the compound from the action of acids, enzymes and other
natural conditions which may inactivate the compound.
Therapeutic compounds of the invention can be formulated to ensure proper
distribution in vivo. For example, the blood-brain barrier (BBB) excludes many highly
hydrophilic compounds. To ensure that the therapeutic compounds of the invention cross
the BBB, they can be formulated, for example, in liposomes. For methods of manufacturing
liposomes, see, e.g., U.S. Patents 4,522,811; 5,374,548; and 5,399,331. The liposomes may
comprise one or more moieties which are selectively transported into specific cells or organs
("targeting moieties"), thus providing targeted drug delivery (see, e.g., Ranade et aL, 1989).
Exemplary targeting moieties include folate or biotin (see, e.g., U.S. Patent 5,416,016 to Low
etaL); mannosides (Umezawaef^/., 1988); antibodies (Bloeman etal, 1995; Owais etal>
1995); surfactant protein A receptor (Briscoe et aL, 1995). In a preferred embodiment,
48
therapeutic compounds of the invention are formulated in liposomes; in a more preferred
embodiment, the liposomes include a targeting moiety.
Delivery and in vivo distribution can also be affected by alteration of an anionic group
of compounds of the invention. For example, anionic groups such as phosphonate or
5 carboxylate can be esterified to provide compounds with desirable pharmocokinetic,
pharmacodynamic, biodistributive, or other properties. Exemplary compounds include IV1
and pharmaceutical^ acceptable salts or esters thereof.
To administer a therapeutic compound by other than parenteral administration, it may
be necessary to coat the compound with, or co-administer the compound with, a material to
10 prevent its inactivation. For example, a therapeutic compound may be administered to a
Q subject in an appropriate carrier, for example, liposomes, or a diluent. Pharmaceutically
,B acceptable diluents include saline and aqueous buffer solutions. Liposomes include water-in-
l gjj oil-in-water CGF emulsions as well as conventional liposomes (Strejan et aL,J. NeuroimmunoL
[i (1984) 7, 27).
j ^15 A therapeutic compound may also be administered parenterally (e.g., intramuscularly,
is* intravenously, intraperitoneal^, intraspinally, or intracerebrally). Dispersions can be
|=y prepared in glycerol, liquid polyethylene glycols, lactose, dextrose and mixtures thereof and in
oils. Under ordinary conditions of storage and use, these preparations may contain a
! fl preservative to prevent the growth of microorganisms. Pharmaceutical compositions suitable
20 for injectable use include sterile aqueous solutions (where water soluble) or dispersions and
sterile powders for the extemporaneous preparation of sterile injectable solutions or
dispersion. In all cases, the composition must be sterile and must be fluid to the extent that
easy syringability exists. It must be stable under the conditions of manufacture and storage
and must be preserved against the contaminating action of microorganisms such as bacteria
25 and fungi. The vehicle can be a solvent or dispersion medium containing, for example, water,
ethanol, polyol (for example, glycerol, propylene glycol, dextrose, and liquid polyethylene
glycol, and the like), suitable mixtures thereof, and vegetable oils. The proper fluidity can be
maintained, for example, by the use of a coating such as lecithin, by the maintenance of the
required particle size in the case of dispersion and by the use of surfactants.
49
Prevention of the action of microorganisms can be achieved by various antibacterial
and antifungal agents, for example, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal,
and the like. In some cases, it will be preferable to include isotonic agents, for example,
sugars, sodium chloride, or polyalcohols such as mannitol and sorbitol, in the composition.
5 Prolonged absorption of the injectable compositions can be brought about by including in the
composition an agent which delays absorption, for example, aluminum monostearate or
gelatin.
Sterile injectable solutions can be prepared by incorporating a therapeutic compound
in the required amount in an appropriate solvent with one or a combination of ingredients
10 enumerated above, as required, followed by filter sterilization. Generally, dispersions are
Q prepared by incorporating the therapeutic compound into a sterile vehicle which contains a
■y
g basic dispersion medium and the required other ingredients from those enumerated above. In
j the case of sterile powders for the preparation of sterile injectable solutions, the preferred
^ methods of preparation are vacuum drying and freeze-drying which yields a powder of the
=AJ15 active ingredient (i.e., the therapeutic compound) plus any additional desired ingredient from
U a previously sterile-filtered solution thereof.
\*i A therapeutic compound can be orally administered, for example, with an inert diluent
: 0 or an assimilable edible carrier. A therapeutic compound and other ingredients may also be
;jy enclosed in a hard or soft shell gelatin capsule, compressed into tablets, or incorporated
20 directly into the subject's diet. For oral therapeutic administration, a therapeutic compound
may be incorporated with excipients and used in the form of ingestible tablets, buccal tablets,
troches, capsules, elixirs, suspensions, syrups, wafers, and the like. The percentage of
therapeutic compound in the compositions and preparations may, of course, be varied. The
amount of therapeutic compound in such therapeutically useful compositions is such that a
25 suitable dosage will be obtained.
It is especially advantageous to formulate parenteral compositions in dosage unit form
for ease of administration and uniformity of dosage. Dosage unit form as used herein refers
to physically discrete units suited as unitary dosages for the subjects to be treated; each unit
containing a predetermined quantity of therapeutic compound calculated to produce the
50
desired therapeutic effect in association with the required pharmaceutical vehicle. The
specification for the dosage unit forms of the invention are dictated by and directly dependent
on (a) the unique characteristics of the therapeutic compound and the particular therapeutic
effect to be achieved, and (b) the limitations inherent in the art of compounding such a
5 therapeutic compound for the treatment of pain and inflammation in subjects, or effecting
sedation.
A therapeutic composition can be administered in time-release or depot form, to
obtain sustained release of a therapeutic compound over time. A therapeutic compound of
the invention can also be administered transdermally (e.g., by providing a therapeutic
10 compound,,with a suitable carrier, in patch form, or in an unguent or cream).
Q Active compounds are administered at a therapeutically effective dosage sufficient to
: JJ mitigate pain and/ or inflammation in a subject. The ability of a compound to mitigate pain
I s ] or inflammation can be evaluated in model systems that may be predictive of analgesia and
~~: anti-inflammation in human diseases, such as animal model systems known in the art
WIS (including, e.g., the preclinical acute pain writhing model in the mouse; and the formalin-
U sensitization model of tissue injury pain in the rat) or by in vitro methods, (including, e.g.,
the assays described above and below, vide infra). The ability of a compound to effect
; D sedation can be evaluated in model systems that may be predictive of sedation of use in
=D treatment of human diseases, such as animal model systems known in the art (including, e.g.,
20 the loss of the righting reflex in the mouse as described, vide infra) or by in vitro methods,
(including, e.g., modulation of the activity of GABA A receptors as described above and below,
vide infra).
It will be appreciated that the ability of a compound of the invention to mitigate pain
and/ or inflammation, in certain embodiments, be evaluated by observation of one or more
25 symptoms or signs associated with pain and inflammation in vivo. Thus, for example, the
ability of a compound to alleviate pain may be associated with an observable improvement in
a clinical manifestation of the underlying pain or inflammation related disease state or
condition, or a slowing or delay in progression of symptoms of the condition. Thus,
monitoring of clinical manifestations of disease can be useful in evaluating the analgesic,
sedative and anti-inflammatory efficacy of a compound of the invention.
Treating or mitigating pain may involve effecting analgesia, effecting sedation,
inhibiting or preventing inflammation, and/or ameliorating the manifestations or impact of
pain inducing stimuli. Modulating a biological process such as the biological levels of cGMP
or cAMP, or activity of soluble GCase, includes regulating increases and decreases in such
activity, and inhibition, potentiation, agonism, or antagonism of the biological process.
Methods of the invention are useful for treating pain and/or inflammation associated
with any disease in which pain or inflammation occurs. Clinically, pain and inflammation
can be associated with, but not limited to, tissue injury, post-operative tissue injury, nerve
injury, post-herpetic neuralgia, phantom limb pain, diabetic neuropathy, arthritis,
dysmenorrhea, endometriosis, cancer, chemotherapy, myocardial infarction, cerebral vascular
occlusion, or result from surgical procedures.
Certain compounds for use in the methods of the invention are commercially
available, whereas others are novel (see hereinbelow and applicants' co-pending application
USSN 09/267,379, filed March 15, 199^). Both types can be synthesized by standard
techniques known in the art. In general, nitrate esters can be prepared from the
corresponding alcohol, oxirane or alkene by standard methods that include: nitration of
alcohols and oxiranes, mixed aqueous/ organic solvents using mixtures of nitric and sulfuric
acid and/or their salts, with temperature control (see Yang etaL, 1996); nitration of alcohols
and oxiranes in acetic anhydride using nitric acid or its salts with or without added acid
catalyst, with temperature control (see, e.g., Louw, etaL, 1976); nitration of an alcohol with a
nitronium salt, e.g., a tetrafluoroborate; nitration of an alkene with thallium nitrate in an
appropriate solvent (Ouellette et al y 197 '6). Compounds of the present invention also can be
prepared as described below.
The contents of all scientific publications and patent documents cited herein are hereby
incorporated herein by reference in their entirety.
The following Examples further illustrate the present invention and are not intended
to be limiting in any respect.
Examples
Example 1: Characterization ofguanylyl cyclase activation
5 Activation of soluble guanylyl cyclase (GCase) by nitrates Illm, IVa, F/b, IVd, IVe,
IVf, IVg, IVj, Va, Vb, and GTN was assayed employing partially purified enzyme freshly
prepared from the 105,000g supernatant fraction of rat aorta homogenates, using the
radioimmunoassay method described by Bennett etal (1992). Dose-response curves were
obtained for GCase activation by nitrates IVa, IVb, IVd, IVe, IVf, IVg, IVj, and GTN in the
10 presence and absence of cysteine and dithiothreitol (DTT; both 2mM). In all cases, data were
normalized to the maximal GTN response carried out in identical GCase preparations.
=3 Experimental incubations were performed at 37°C for 10 min. The data for IVd is
:s=S
summarized in Fig. 1. The GCase assay data show that IVd activates GCase, with a
.[1 submillimolar EC-50 (effective concentration for 50% of the subjects) in the absence of any
1^15 added thiol, in contrast to GTN, which requires added cysteine. Compounds IVd also
activates GCase in the presence of DTT, in contrast to GTN, which, enigmatically, does not.
jij Relative to GTN itself, a wide range of potency was observed for these novel nitrate esters.
; * No activation of GCase by glycerol mononitrates was observed in this assay at the
0 concentrations of nitrate employed.
; t i
' ssr
20 Activation of GCase, in vitro, by some of these organic nitrates is via release of NO,
since in the presence of cysteine, substantial NO is released at rates which are measurable
amperometrically, using the method described by Artz and Thatcher (1998). By comparison,
nitroglycerin, which is currently understood by others skilled in the art to act only as an NO-
donor therapeutic agent, does not release NO at a rate measurable amperometrically, in the
25 absence nor the presence of cysteine. Relative rates for NO release, at 37°C, pH 7.4, in the
presence of cysteine (2mM) from nitrates Vj, Vu, Vi, Vh, Vg, Vf, and Ve, (ImM) were 1.0, 1.0,
1.8, 0, 1.2, 0.5, 1.8, respectively. Thus modification of the structure of these organic nitrates
can be used to control their NO-releasing ability and also to modulate GCase activity.
To test for potential differences in GCase activation by nitrates, the effects of Illm,
30 IVh, Va, Vb, and GTN were assayed in brain and vascular tissue. IVh had no effect on GCase
activity in either rat aorta or rat hippocampus (Fig. 2). Illm had greater efficacy to stimulate
GCase activity compared to GTN in both rat aorta and rat hippocampus (Fig. 2). Vb was
found to be equivalent to GTN in efficacy and potency for activation of GCase in both rat
aorta and rat hippocampus (Fig. 3). Va was found to have greater efficacy, but equal potency,
to GTN in rat aorta (Fig. 3a). In contrast, Va had greater efficacy and greater potency to
stimulate GCase in rat hippocampus (Fig. 3b). These data illustrate that nitrates have
differential effects on GCase activation that are dependent on both structure of the compound
and the tissue assayed for GCase activity, supporting the notion that effects of nitrates elicited
through GCase activation, such as analgesia and vasodilation, are separable and may be
regulated in a tissue-specific and/or activity-specific manner, by appropriate choice of organic
nitrate.
As further examples of the potential for modulating potency, efficacy and tissue
selectivity for activation of GCase, by choice of an appropriate organic nitrate, nitrates Vaa
and Vt were assayed in brain and vascular tissue. In the presence (+) and absence (-) of ImM
cysteine, the potency (EC-50 values) and efficacy (maximal activation) were measured for
activation of GCase from rat hippocampus (Table 2).
Table 2
ImM
cysteine
EC-50 (hipp.),
M
EC-50 (aorta),
M
maximal (hipp.),
relative to
GTN"
maximal (aorta),
relative to
GTN"
Vaa
+
1.4 x 10 4
1.8 x 10- 4
5.1
2.2
Vaa
5.7 xlO' 5
1.8 x lO 4
2.1
1.0
Vt
+
5.1 x 10" 5
6.5 x lO" 5
3.0
2.3
Vt
1.7 x 10- 5
1.8 x 10*
1.8
1.7
a) Normalized to GTN + ImM cysteine maximal response.
Example 2: Characterization of cyclic GMP accumulation
In order to extend the GCase data further, the effects of nitrates Va, Him, Vb, Vc, and
IVk on cyclic GMP accumulation in intact isolated rat aorta were examined (Figs. 4, 5).
Thoracic aortic strips were prepared from male Sprague-Dawley rats (Charles-River, Canada)
as described in McGuire etal. (1994) and Stewart etal (1989). Tissues were contracted
submaximally with phenylephrine (O.ljxM) and exposed to various concentrations of drug for
1 min. Cyclic GMP accumulation was determined using the radioimmunoassay method
described by Bennett etal (1992). At concentrations of 1 |uM and 10 |iM, GTN and IVk
significantly increased cGMP accumulation (Figs. 5). At a concentration of 1 |LiM, Va, Elm,
Vb, and Vc did not significantly increase cyclic GMP accumulation (Figs. 4a, 5a). At a
concentration of 10 jxM, Va, Vb, and IVk significantly increased cyclic GMP accumulation
whereas Him and Vc did not (Figs. 4b, 5b).
Sections of rat hippocampus (400 jam) were prepared and incubated in oxygenated
Krebs solution at 37°C. After a 60-min equilibration period, the brain slices were stimulated
with different concentrations of Va or GTN for 3-min. Cyclic GMP accumulation was
determined as described above for aortic strips. Figure 6 shows that Va causes a
concentration-dependent increase in the tissue levels of cGMP in rat hippocampal brain slices
in vitro, and that at high concentration (100 |xM) Va is more effective than GTN in elevating
cGMP levels in hippocampal brain slices in vitro. These data are in very good agreement with
the differential effects of Va and GTN on hippocampal GCase activity shown in Figure 3b.
Example 3: Characterization of relaxation of isolated blood vessels
In order to extend the GCase data, the relaxing effects of nitrates Him, IVc, IVd, IVf,
IVg, IVh, IVk, Va, Vb, and Vc on rat aortic tissue were examined. Thoracic aortic strips were
prepared from male Sprague-Dawley rats (Charles-River, Canada) as described in McGuire et
al (1994), and Stewart etal (1989). Tissues were contracted submaximally with
phenylephrine (0.1 nM) and exposed to various concentrations of nitro vasodilator to obtain
concentration-response curves. In this intact tissue assay, all of the nitrates were observed to
cause relaxation of the tissue with a maximal relaxant response equal to that obtained with
GTN. However, the compounds differed in potency, with EC-50 values of 7.87 nM, 94.3 nM,
6.59 nM, 25.2 \M, 11.0 |iM, and 0.203 \M, for GTN and compounds Va, IVd, IVg, IVf, and
IVc, respectively (Fig. 7,8). In another series of experiments, the EC-50 values for relaxation
were 0.61 nM, 3.19 nM, 8.40 nM, 0.153 jiM, 0.437 nM and 6.89 ^M for GTN, IVk, Vb, Him,
Vc, and IVh, respectively (Fig. 7,8). Compounds IVd and IVc were tested for their ability to
cause vascular relaxation in tissues that had been made tolerant to the relaxant effect of GTN.
GTN tolerance was induced by incubating tissues with high concentrations of GTN (0.5mM
GTN for 30 min). Under these conditions, the maximal relaxant effects of IVd (Fig. 12a) and
IVc (Fig. 12b) were not significantly different to that of untreated tissue. The EC-50 for
relaxation was increased approximately threefold, but the difference was not statistically
significant.
Example 4: Characterization of blood pressure changes in the whole animal
To test for differential effects of nitrates on blood pressure responses, Va and GTN
were injected into rats in which the abdominal aorta was cannulated for blood pressure
recording. In the first experiment, Va and GTN were injected subcutaneously at a dose of 400
|amol/kg body weight into conscious, freely moving animals. GTN caused a small and
transient decrease in blood pressure in these animals, whereas Va had no discernable effect on
56
arterial blood pressure (Fig. 9). Va and GTN were subsequently tested in anesthetized rats in
which the abdominal vena cava was also cannulated to allow for bolus intravenous injection of
drugs. In this preparation, GTN caused a substantial and dose-dependent decrease in arterial
blood pressure. In contrast, Va at equal doses had very modest effects on blood pressure at
5 doses lower than 2 jimol/kg body weight (Fig. 10). These data are in very good agreement
with the results obtained for these two agents using the isolated blood vessel preparation.
The plasma levels of nitrates Vb and Vc (the denitrated metabolite of Vb) were
measured to gain insight into the handling of these molecules in the body. Cannulas were
placed in the abdominal aorta for blood sampling. After a two-day recovery period, a single
10 subcutaneous dose of Vb (200 |imol/kg) was administered and blood samples collected over a
'3 period of six hours. Samples were centrifuged, the plasma collected, and the concentration of
: - S
'saF
: p Vb and Vc determined by gas-liquid chromatography by the method of McDonald and
I j Bennett (1990). The data obtained for Vb and Vc indicate that nitrates achieve maximal
1 2 plasma levels within 30 minutes after subcutaneous injection, and therafter decline at a steady
;aJ15 rate (Fig. 11). These data suggest that nitrates have excellent bioavailability after subcutaneous
injection.
iU
ill
'2 Example 5: Characterization of the analgesic effects of novel organic nitrates in a model of acute
; 3 pain
20 Injection of dilute acetic solutions into the peritoneum of a mouse induces writhing
movements that can be quantified. We adopted the methodology described by Bak etal
(1998) to test for analgesic effects of organic nitrates in this mouse model. Each mouse was
given an intraperitoneal injection of 0.5 mL of a 0.6% solution of acetic acid in distilled water.
After a 5 minute delay, the number of writhing movements was counted over a 10 minute
25 period. To test the efficacy of novel organic nitrates in this model, drugs were administered at
doses of 100-500 mg/kg (given by subcutaneous injection) 15 minutes before the
intraperitoneal injection of acetic acid. In this model of acute pain, Vm induced a significant,
dose-dependent analgesic effect, manifested as a decrease in the number of writhes per 10
minute period after intraperitoneal injection of dilute acetic acid (Figure 13a). Va was also
4
57
able to act as an analgesic (decreased writhing) in this experimental model when administered
at a dose of 500 mg/kg subcutaneously (Figure 13b).
Example 6: Characterization of the analgesic effects of novel organic nitrates in a model of
5 byperalgesia/allodynia
Under light halothane anesthesia, male Sprague-Dawley rats were injected
subcutaneously with 0.05 mL of 5% formalin into the dorsal surface of one hind paw as
described in Malmberg and Yaksh (Anesthesiology (1993) 79, 270-281). The number of
spontaneous paw flinches was determined in 1 minute blocks at 5 minute intervals for 60
10 minutes. Formalin injection in the paw produces two distinct phases of pain; an acute phase
□ occurring within the first 5-10 minutes, and a delayed phase that develops between 15-30
J minutes after formalin injection. The acute phase of the pain response to formalin is caused
by activation of peripheral nociceptive sensory afferents (C-fibres) by the peripheral stimulus.
'H The delayed pain response is considered to be a hyperalgesia/ allodynia caused by a
l^jlS combination of sensitization of peripheral sensory afferents and sensitization of synaptic
|^ connections in the spinal cord. The formalin test in the rat is considered to be an appropriate
\j£ model for tissue injury pain occurring in humans (Tjolsen etaL 9 Pain (1992) 51, 5-17; Yaksh,
iU
: -Q TIPS (1999) 20, 329-337). In this experimental model of inflammatory tissue injury pain, Vm
:Q (500 mg/kg) significantly reduced both phases of the pain response to formalin injection (Fig.
20 14a,b).
Example 7: Synthesis ofllle
To acetic anhydride (3 mL) was added gradually, with stirring, 70% nitric acid (0.26
mL), while keeping the temperature between 20-30° by external cooling. With continuous
25 vigorous stirring the mixture was cooled to -30-35° and 2',3'-dideoxy-3-thiocytosine (0.25 g)
was added. After 10 min at -35°, the reaction mixture was heated up to -20° and then stirred
at -20-10° for 15 min and 10 min at 0°. The resulting reaction mixture was poured into ice-
water, stirred for 1 h, then NaHC0 3 was added by portions until C0 2 evolution ceased. The
water solution was extracted with 3x20 mL of ethyl acetate. Combined extracts were dried
(MgS0 4 ) and concentrated. 0.38 g of slightly yellowish oil was obtained. The oil crystallized
in a day and was recrystallized from CHC1 3 . Yield 52%. Conversion to the nitrate was
evidenced by the significant downfield shift of the C5' proton multiplet from 8 3.6 to 4.85
ppm.
Example 8: Synthesis of nitrate Illf
0.26 mL (4.15 mmol) cone. HN0 3 was added to 2 mL acetic anhydride such that the
temperature did not exceed 25 - 30 °C The mixture was cooled at 0 - 5 °C and 0.3 g (1.88
mmol) of 5-(l,2-dihydroxyethyl)-4-methylthiazole was added in several portions, the
temperature being kept below 5 °C. The reaction mixture was stirred at 0 - 5 °C for 45 min
and then 0.45 mL water was added. The mixture was stirred for 30 min and then rotavary
evaporated. The residue was neutralized by adding 5 mL of saturated NaHC0 3 solution and
the organic product was extracted with ethyl acetate. The organic layer was concentrated and
the dinitrate Illf was purified through column chromatography (silica gel/ ethyl acetate
eluant). A slightly yellow solid was obtained. Yield: 0.150 g (32 %).
Example 9: Synthesis of nitrate Illi
Nitrate Illi was obtained by two routes. Route I proceeded from the elimination
reaction of Him in basic solution. Route II proceeded from nitration of £ra?zs-3-bromo-4-
hydroxytetrahydrothiophene-l,l-dioxide, yielding nitrate Illn, followed by reaction with a
weak base, e.g., sodium thiocyanate in 2-butanone. Purification may be achieved with silica
flash column chromatography using 1:1 hexane:ethyl acetate as eluant.
Example 10: Synthesis of nitrate III]
l,4-Dibromo-2,3-butanediol may be nitrated: (a) using a nitration mixture prepared
from HN0 3 and H 2 S0 4 over 2 days; or (b) using acetyl nitrate reacting for 2 hours. Work-up
requires quenching of the reaction mixture in ice-water for an hour, extraction, drying, and
evaporation. Successful purification of the title compound by silica gel column
59
chromatography is achieved on a 25 g scale using a mixture of 70% hexane and 30% CH 2 C1 2 as
eluent
Example 11: Synthesis of nitrate Ve
5 4-Methylbenzenethiol was obtained by adaptation of literature procedures from p-
toluidine 0.-P. Morizur, Bull. Soc. Cbim. Fr. (1964) 1338-1342; Bourgeois, Red Trav. Cbim.
Pays-Bas (1899) 18, 445-450). p-Toluidine hydrochloride (14.2 g, 0.098 mole) was diazotised at
5°C with concentrated hydrocloric acid (16.5 mL) and sodium nitrite (7.2 g, 0.104 mole) in
water (12 mL). The solution of diazonium salt was added over 1.5 h to a solution of ethyl
10 xanthate (24 g, 0.149 mole) in water (30 mL) at 45-50°C. The mixture was kept at this
;3 temperature, under stirring, for a further 1 h. The xanthate ester was separated as a maroon
g oil, washed with 50 mL 10 % NaOH and with water to neutral pH and dried over MgS0 4
A (20g of crude product). The crude xanthate was dissolved in 60 mL absolute ethanol and to
'^4 this solution 20 g KOH (pellets) were added in portions. The reaction mixture was refluxed
yl5 under stirring and Ar for 8 h, then concentrated under vacuum. The concentrate was taken
u up in 50 mL H 2 0 and extracted with 3x100 mL diethyl ether. The aqueous layer was acidified
; * with a 6N H 2 S0 4 solution and extracted with 3x100 mL CH 2 C1 2 . The combined extracts
! D were washed with water, dried over MgS0 4 , evaporated and flash columned on silica gel,
; y
:£) eluant hexanesiethyl acetate =9:1, giving 10 g (81.56 %) of 4-methylbenzenethiol. 1 H-
20 NMR(CDC1 3 , 300MHz):7.18-7.24 (m, arom 2H), 7.04-7.11 (d, arom 2H, J 7.93), 3.41(s, 1H),
2.32(s, 3H). 13 C-NMR(75.48 MHz): 21.34, 128.95, 130.24, 130.29, 136.05.
The dinitrate IVd (9.67 mmoles) was dissolved in 10 mL distilled water and the
solution kept under Ar for 30 minutes. To this solution, a solution of 0.8 g (6.46 mmoles) of
4-methylbenzenethiol and 7 mL 1M NaOH was added dropwise. The resulting emulsion was
25 stirred for 15 min and then extracted with 3x20 mL CH 2 C1 2 . The combined organic extracts
were washed with H 2 0, dried over MgS0 4 and concentrated under vacuum. The remaining
oil was purified by flash column chromatography on silica gel, eluant hexanes: ethyl
acetate=9:l, giving the product Ve (1.097 g, 52.22 %). 'H-NMR^DClj, 300MHz):7.44-7.51
(m, arom 2H), 7.17-7.24 (d, arom 2H, J 7.91), 5.47-5.59 (m, 1H), 4.83-4.93 (dd, 1H, J 12.81,
60
278), 4.57-4.67 (dd, 1H, J 12.82, 5.71), 3.02-3.12 (dd, 1H, J 14.48, 6.01), 2.9-2.99 (dd, 1H, J
14.47, 7.72), 2.38 (s, 3H). 13 C-NMR(75.48 MHz): 21.53, 36.78, 69.82, 77.68, 130.52, 130.62,
132.55, 139.23.
5 Example 12: Synthesis of nitrate Him
3,4-Epoxytetrahydrothiophene-l,l-dioxide (250 mg,1.9 mmol) was refluxed for 24 h in
10 mL of water and 25mg of toluenesulfonic acid. After the first 6 h, another 25 mg of the
acid was added. The reaction was monitored by thin layer chromatography (TLC) (5%
methanol in dichloromethane). Purification was by Si flash column chromatography using
10 5% methanol/CH 2 Cl 2 as eluent to afford 200 mg of diol. The diol was nitrated in a cooled
3 solution of cone, sulfuric acid (2 mol eq.), nitric acid (70%, 2 mol eq.) in an ice bath. The
2 temperature was maintained as close to 0°C as possible. The ice bath was removed and the
mixture was allowed to stir for 1 hour (reaction was monitored by TLC, 100% CH 2 C1 2
'4 eluent). The acid layer was removed and the organic layer washed with: (i) water; (ii) 10%
jjl5 sodium carbonate; (iii) 10% urea; (iv) water. Drying over sodium sulfate, filtration and
^ concentration, yielded crude product which was purified by flash column chromatography,
* with dichloromethane as eluent. An alternative route involves direct nitration of 3,4-
fl epoxytetrahydrothiophene-l,l-dioxide in a similar nitration mixture.
0
20 Example 13: Synthesis of nitrate IVk
1.17 mL (18.2 mmol) concentrated HN0 3 was added, under stirring and cooling (0-5
°C), to 1 mL (18.2 mmol) concentrated H 2 S0 4 and then 2 g (14 mmol) of 4-methyl-5-(2-
hydroxyethyl)thiazole was added dropwise into the nitration mixture, the temperature being
kept under 10 °C. The mixture was stirred for 3 hours at room temperature, diluted with 10
25 mL of water and neutralized with solid NaHC0 3 . The organic product was extracted with
ethyl acetate and purified by column chromatography (silica gel/ ethyl acetate eluant) to
produce a colorless oily product. Yield: 1.18 g (45%).
61
Example 14: Synthesis of nitrate IVi
0.03 g (0.035 mL) of allyl cyanide was added to a stirred suspension of 0.22 g (0.5
mmol) of Tl (N0 3 ) 3 .3H 2 0 in 2 mL of pentane. After 20 min of vigorous stirring the pentane
solution was decanted and evaporated to dryness. After evaporation the residual oil (0.44 g)
5 was columned (CH 2 Cl 2) Rf 0.64 (CHjCy. Clean oil immediately crystallized during attempt
to dissolve it in CDC1 3 . Yield 0.065 g (76%). The structure of IVn was confirmed by X-ray
analysis. IR (film): 1297.03, 1678.91, 2258.91 (CN). Mass spec, m/z (defragment, %): 191.9
(M+H, 2.44), 129.0 (16.41), 81.9 (100). Calculated for C 4 H 5 N 3 0 6 191.02.
10 Example 15: Synthesis of nitrate IVm
□ 0.9 g (0.75 mL, 4.92 mmol) of allyphenyl sulfone was added dropwise to a stirred
i £ E
3 suspension of 2.43 g (5.47 mmol) of Tl (N0 3 ) 3 .3H 2 0 in 10 mL of pentane. The resulting
^ mixture was stirred overnight. The pentane solution was decanted. 2x10 mL of methanol
' u 4 were added to the reaction mixture, stirred for 10 minutes and extracts were added to the
!x|15 pentane solution. The combined extracts evaporated to dryness and purified by silica flash
u column chromatography using CH 2 Cl 2 as eluant Yield 0.08 g (15%). IR (KBr): 1152.39,
|}j 1290.91, 1273.12, 1353.83, 1646.08. Mass spec, m/z (defragment, %): 307.0 (M+ 1, 66.5),
i
*£j 244.0 (100%). Calculated for C 9 H 10 N 2 O 8 S 306.02.
■U
l Q
20 Example 16: Synthesis of nitrate Va
2.2 g (7.3 mmol) of nitrate IVd was dissolved in 5 g of cold H 2 0 2 (30%, 0°C) and then
1 g of 10 % H 2 S0 4 was added. The mixture was stirred at 0-5 °C until a white oil separated
(ca. 30-60 min). The aqueous layer was discarded and the oil was dissolved in
dichloromethane, washed successively with water, then NaHC0 3 solution and finally water.
25 The organic solution was dried over MgS0 4 . Removal of the solvent produced 1.3 g of the
crude product which was purified by column chromatography (Silicagel, CH 2 Cl 2 /hexanes :
70/30). Yield: 0.650 g (45 %).
62
Example 17: Synthesis of nitrate Vc
3 g (8.88 mmol) of l,4-dibromo-2,3-dinitrobutanediol and 2.81 g (18 mmol) of
Na 2 S 2 0 3 .5H 2 0 were dissolved in the mixture of 100 mL of methanol and 45 mL of H 2 0. The
resulting solution was heated during 4 days at 40-45°. After this time the reaction mixture was
5 partially evaporated to reduce the volume of solvents. The resulting mixture was extracted
4x50 mL of ethyl ether. The extracts were combined, washed (H 2 0), dried (MgS0 4 ) and
evaporated to minimum. Column chromatography afforded the title compound in 10% yield,
seperated from Vb the major product.
10 Example 18: Synthesis of nitrate Vy
□ The title compound was synthesised by the reaction of IVd with 4-methyl-5-thiazole
ijLJ
g ethanthiol, in a similar procedure to that used in Example 11. The crude product was purified
: ~* by column chromatography (Silicagel, ethyl acetate eluant) to give product (50 mg, 27.32
^ %). 1 H-NMR(CDC1 3 , 300MHz): 8.22 (s, 1H), 5.49-5.6 (m, 1H), 4.9-5.00 (dd,lH), 4.64-478 (dd,
\d5 1H), 3.14-3.22 (t, 2H), 2.89-3.07 (m, 4H), 2.45 (s, 3H). Mass spec, m/z (EI + , fragment, %):
u 355.0 .Calculated for C 9 H 13 N 3 0 6 S 3 : 355.0. The 4-Methyl-5-thiazole precursor was obtained
: ^ from 4-methyl-5-thiazole ethanol, thiourea and hydrobromic acid by adaptation of literature
: y
; B procedures (R.L. Frank, P. V. Smith,/. Am. Chem. Soc. (1946) 68, 2103-2104). A mixture of 2
•Q g 4-methyl-5-thiazole ethanol (13.965 mmoles), 1.063 g(13.965 mmoles) thiourea and 9.45 g
20 (56 mmoles) hydrogen bromide as 48 % hydrobromic acid was refluxed for 7 h with stirring,
under Ar. A solution of 2.24 g (56 mmoles) of NaOH in 20 mL of water was then added and
the mixture was refluxed without stirring for 2 h. The layers were separated, and the acidified
aqueous layer was extracted with three 30 mL portions of CH 2 C1 2 . The extracts and original
organic layer were combined, dried over MgS0 4 and concentrated under vacuum to afford 1.9
25 g crude product which was purified by column chromatography using ethyl acetate as eluent
(Yield 1.6 g, 75%). 'H-NMRtCDCl,, 300MHz): 8.5 (s, 1H), 2.94-3.02 (t, 2H),2.62-2.72 (q, 2H),
2.33 (s, 3H), 1.38-1.45 (t, 1H). 13 C-NMR(75.48 MHz): 15.41, 26.41, 31.51, 39.72, 129.36,
149.90
•
63
Example 19: Synthesis of nitrate Illk and IIIl
Synthesis from dinitrate III] proceeded by refluxing with sodium ot potassium
thiocyanate (2 eq.) in 2-butanone for 8 h. After cooling, a precipitate was removed by
filtration and the filtrate was concentrated. Nitrates Illk and IIIl were separated by silica flash
5 column chromatography with hexane/dichloromethane as eluent.
Example 20: Synthesis of nitrate Vk
IVd ( 0.43 g, 1.37 mmol) was dissolved in 10 mL of distilled water and the emulsion of
0.23 g (1.28 mmol) of the ethyl ester of thiosalycilic acid in 1.3 mL of 1 M NaOH was added.
10 The resulting solution immediately became turbid and was stirred for 3 min, then extracted
i3 with ethyl acetate (4x15 mL). The resulting extracts were washed with H 2 0, dried (MgS0 4 ),
j= and concentrated by evaporation. The residue was flash columned on silica gel, eluant
^ hexane:ethyl acetate=9:l (R, 0.23), giving 0.27 g (55%) of Vk. l H-NMR(CDC\ } , 300 MHz):
^ 8.06-8.12 (IH, dd, J 8.12, 0.36), 7.99-8.04 (IH, dd, J 7.8, 1.15), 7.51-7.59 (IH, m, J 7.24, 1.44),
i J15 7.21-7.29 (IH, m, J 7.35, 0.54), 5.40-5.49 (IH, m), 4.70-4.78 (IH, dd, J 13.04, 2.95), 4.33-4.45
u (3H, m, superposition of dd 0 13.08, 6.01) of IH from CH 2 -ON0 2 and quart, from Q- CH 2 -
|}j CH 3 ), 2.66-2.87 (2H, m), 2.06-2.15 (2H, quart., J 6.92), 1.35-1.43 (3H, t, J 7.14). "C-NMR:
; D (CDC1 3 , 75.48 MHz): 166.21, 140.41, 132.78, 131.46, 127.63, 125.48, 77.25, 71.03, 61.42, 32.51,
if)
.Q 28.29, 14.17. Mass spec, m/z (EI + , fragment, %): 392.3 (M + , 3.69), 153 (100). Calculated for
20 C 13 H 16 N 2 0 8 S 2 392.03.
Example 21: Chiral synthesis of nitrate IVd
A 50 mL round-bottom flask, equipped with a magnetic stirrer, was charged with 10
mL of tot-butyl alcohol, 10 mL of water, and 2.8 g of a catalyst (AD-mix-fi, Aldrich:
25 K.B.Sharpless, W.Amberg, Y.L.Bennani, G.A.Crispino, J.Hartung, K.-SJeong, H.-L.Kwong,
K.Morikawa, Z.-M.Wang, D.Xu, X.-L.Zhang, /. Org. Chem. (1992) 57, 2768-2771). Stirring at
room temperature produced two clear phases; the lower aqueous phase appears bright yellow.
The mixture was cooled to 4° and 0.2 mL (2 mmol) of allylbromide was added at once, and the
heterogeneous slurry was stirred vigorously at 4-5° for 2.5 h (monitoring by TLC
64
hexane:methanol= 1:9). While the mixture was stirred at 0°C , solid sodium sulfite (3 g) was
added and the mixture was allowed to warm to room temperature and stirred for 1 h. Then
20 mL of ethyl acetate was added to the reaction mixture, and after separation of the layers,
the aqueous phase was further extracted with ethyl acetate. The combined organic extracts
5 were dried (MgS0 4 ), concentrated in vacuo and purified by flash chromatography on silica
(hexane:methanol= 1:9, R f 0.5), to yield chiral l-bromo-2,3-propanediol. Yield 0.2 g (55.5%).
Optical rotation: minus. Nitration to l-bromo-2,3-dinitroxypropane was achieved using
reaction in HN0 3 /H 2 S0 4 (K. Yang, J.D. Artz, J. Lock, C. Sanchez, B.M. Bennett, A.B.
Fraser, G.R.J. Thatcher, J.Ckem.Soc, Perkin Trans. (1996) 1, 1073-1075) and the product was
10 purified by flash chromatography on silica gel (hexane:CH 2 Cl 2 =2:3, Rf 0.5). Yield 55.5%.
q Optical rotation: minus. The dinitrate IVd was synthesised from chiral l-bromo-2,3-
| propanediol by our usual procedure (K. Yang, J.D. Artz, J. Lock, C. Sanchez, B.M. Bennett,
^ A.B. Fraser, G.R.J. Thatcher, J.Cbem.Soc, Perkin Trans. (1996) 1, 1073-1075). and purified by
flash chromatography on silica (ethyl acetate:methanol=9:l). Yield 2.27%. Optical rotation:
ylS plus.
pj Example 22: Chiral synthesis of nitrate Vk
\q The same procedure as described above for racemic Vk was utilized for the
: stereospecific synthesis of Vk from the sterechemically resolved, chiral dinitrate IVd.
20
Example 23: Synthesis of tetranitrate Vx
The title compound was synthesised by the reaction of IVd (0.345g, 1.15 mmoles) with
captopril methyl ester (0.2 g, 0.865 mmoles) in the presence of 1 mL of 1M NaOH. The crude
product was purified by column chromatography (Silicagel, ethyl acetate) to afford 0.1 g
25 (18.03 %) of unsymmetrical disulphide/H-NMR^DC^, 300MHz): 5.49-5.61 (m, 1H), 4.88-
4.90 (m, 1H), 4.62-4.74 (m, lH),4.40-4.55 (m, lH),3.59-3.78 (m, superpos. 5H), 2.69-3.21 (m,
superpos. 5H), 2.14-2.29 (m, 1H), 1.90-2.12 (m, 3H), 1.22-1.28 (d, 3H). 13 C-NMR(75.48 MHz):
a mixture of enantiomers can be seen: 17.39, 25.20, 29.40, 36.41, 36.51, 38.55, 41.94, 42.19,
47.32, 52.56, 59.12, 69.90, 70.29,77.89, 78.00, 173.01, 173.48.
t
65
Example 24: Synthesis of nitrate Vq
The title compound was synthesised by the reaction of Bunte salt (2.4 g, 8 mmoles)
with 4,4'-thiobisbenzenethiol (0.5 g, 2 mmoles) in the presence of 4.4 mL of 1M NaOH The
5 crude product was purified by column chromatography (silica gel, hexanes/CH A : 3/7) to
afford 0.28 g (21.82 %) disulphide. 'H-NMR(CDC1, 300MH 2 ): 7.2-7.6 (m, arom 8H), 5 42-
5.56 (m, 2H), 4.82-4.95 (dd, 2H), 4.55- 4.67 (dd, 2H), 2.93-3.15 (m, 4H).
Example 25: Synthesis of nitrate Vr
10 The title compound was synthesised by the reaction of IVd (0.5 g, 1.58 mmoles) with
g ethyl 2-mercapto-3-(3',4'-methyl e nedioxy-phenyl) propenoate (0.2 g, 0.8 mmoles). The crude
| product was purified by column chromatography (Silicagel, hexanes/CH 2 Cl 2 : 3/7) to give Vr
l3 {0A 8)28 -° 9 %) ' '^(CDCl, 300MH 2 ):7.97 (s, IH), 7.48-7.51 (d, IH), 7.17-7 22 (m IH)
J 6 " 84 - 6 - 89 {d ' 1H) ' M5 2H )> 5 - 48 - 5 - 5 %> 1H),4.814.89 (dd, IH), 4.53-4.61 (dd, IH), 4 25-4 4
w 15 (m, 2H), 3.05-3.10 (m, 2H), 1.35-1.45 (t, 3H). »C-NMR(75.48 MHz): 14.17, 36.27, 62 15
I 69.71, 76.57, 101.77, 108.27, 110.31, 125.20, 127.55, 127.87, 146.87, 147.811, 149.76, 165.89.
| Ethyl 2-mercapto^yA'-methylemdioxy.pheny^ was obtained by the
; ^ following route:
0 (1) Ethylisothiocyanate (10 g, 0.115 moles), 7.9 g(0.086 moles) mercaptoacetic acid and 5
20 mL pyridine in C 6 H 6 was refluxed, cooled and filtered yielding 3-ethylrhodanine, which was
used m the next step without any further purification. Reaction of 3-ethylrodanine with
piperonal in the presence of sodium acetate was performed in CH 3 OH under refluxing for 1
h. The yellow precipitate obtained after cooling and filtration, was washed several times with
CH 3 OH on the filter and crystallizated from CH 3 OH to give 3-ethyl-5-piperilidenerodanine
25 - 'H-NMRPMSCH, 300MHz):7.72 (s, IH), 7.1-7.3 (m, 3H), 6.14 (s, 2H), 3.95-4.15( q , 2H)
U5-1.20 (t, 3H). »C-NMR(75.48 MHz): 12.76, 40.28, 103.07, 110.21, 110.47, 120.68, 127.89,
128.07, 134.01, 149.23, 150.74, 167.57, 193.68.
(2) 3-Ethyl-5-pi per ilidene- rodanine (Ig, 3.4 mmoles) was added to a stirred solution of
0.16 g (6.8 mmoles) in 8 mL abs. ethanol and the mixture was refluxed for 30 min. To the
30 solution cooled to room temperature 5 mL H 2 0 were added and the mixture was hydrolized
66
with 10% HC1 and extracted with ether. The ether phase was separated, dried (MgS0 4 ) and
evaporated to an oil. Since the investigation by TLC of the crude reaction mixture indicated
the presence of piperonal, the reaction mixture was dissolved in CH 2 C1 2 and the obtained
solution was washed with 1M NaOH. The aqueous phase was extracted twice with CH 2 C1 2
5 and then neutralized with dilute HCL Free thiol was extracted with ethyl ether. After
concentration, the product was purified by column cromatography eluating with
hexane/ethyl ether:8/2. Yield 0.4 g (62.4 %). 'H-NMRtCDC^, 300MHz):7.69 (s, 1H), 7.7.25-
7.29 (d, 1H, J 1.46), 7.17.18 (m, 1H), 6.85-6.9 (d, 1H, J 8.13), 6.01 (s, 2H), 4.75 (s, 1H), 4.28-
4.38 (q, 2H, J 7.12 ), 1.35- 1.42 (t, 3H, J 7.13). 13 C-NMR(75.48 MHz): 14.20, 62.5, 101.41,
10 108.37, 109.43, 121.06, 125.45, 129.20, 134.68, 147.80, 148.05, 165.43.
5 Example 26: Synthesis of nitrate IVs
The title compound was synthesised by the reaction of IVb with dithiothreitol in
H CH 3 OH and was isolated as an oil in 15-20% yield (CAUTION: stench). 'H-NMRfCDClj,
U5 300 MHz): 5.23-5.32 (1H, m), 4.87 (1H, dd, J 12.82, 3.22), 4.68 (1H, dd, J 12.83, 6.09), 2.77-
l u 2.94 (2H, m), 1.66 (1H, t, J 9.07). 13 C-NMR: (CDC1 3 , 75.48 MHz): 79.39, 69.30, 23.68.
!U
llj
; S Example 27: Synthesis of nitrate IVt
; n The title compound was synthesised by the reaction of nitrate IVs with acetyl chloride
20 in CHClj. Isolated yield 50%. 'H-NMR^DCl,, 300 MHz): 5.29-5.38 (1H, m), 4.76 (1H, dd, J
12.94, 3.11), 4.55 (1H, dd, J 12.94, 6.37), 3.30 (1H, dd, J 14.06, 5.98), 3.13 (1H, dd, J 14.61,
6.35). "C-NMR: (CDC1 3 , 75.48 MHz): 194.10, 77.00, 69.79, 30.42, 27.78. Mass spec, m/z
(EI + , fragment, %): 240.0 (M + , 1.17), 193.9 (M-N0 2 , 10.86), 148.8 (100). Calculated for
C 5 H 8 N 2 0 7 S 240.01 .
25
Example 28: Synthesis of nitrate IIIw
Diethyl l-chloro-2-trimethylsiloxypropylphosphonate was obtained by adaptation of
literature methods (T.Azuhata, Y.Okamoto, Synthesis (1983) 916-917). This phosphonate was
quantitatively converted to diethyl l-chloro-2-hydroxypropylphosphonate using CH 3 OH.
#
67
After stirring for 15 min the resulting reaction mixture was evaporated to a minimum and
subjected to nitration with a mixture of HN0 3 and H 2 SO, Work-up and flash column
chromatography on silica (ethyl acetate eluant) yielded pure product in 25% yield. »P
(CDCl, 162 MHz): 24.60. >H (CDC1 3 , 300 MHz): 5.31-5.45 (m, 1H), 3.92-4.08 (m, 4H), 3.63-
5 3.81 (m, 2H), 2.03-2.30 (m, 2H), 1.16-1.24 (superposition of 2 t, 6H, J 7). 13 C (CDC1 3 75
MHz): 76.83, 62.15 (d, J 6.37), 43.77 (d, J 8.95), 27.08 (d, J 142.00), 15.99 (d, J 5.88).
Example 29: Synthesis of nitrate IIIx
Treatment of IIIw with 1 mole of Me 3 SiBr for 1 h with subsequent addition of
10 CH 3 OH provided the monodealkylated phosphonic acid in high purity. Transformation of
the free acid to its sodium salt IIIx was achieved using the cation-exchange resin Amberlite IR-
122-Na* form. »P (CD 3 OD, 122 MHz): 17.62. >H (CD 3 OD 300 MHz): 5.38-5.63 (m, 1H),
3.75-4.25 (superposition of 2 m, 4H), 1.88-2.20 (m, 2H), 1.12-1.28 (t, 3H). »C (CD 3 OD 75
MHz): 81.14, 61.17 (d, J 5.41), 45.56 (d, j 5.94), 29.35 (d, J 131.74), 17.00 (d, J 6.75)
15
Example 30:
The effect of IVk was tested in Xenopus oocytes expressing human recombinant
GABA A receptors by the two-electrode voltage clamp technique as described in Reynolds and
Maitra {European Journal of Pharmacology (1996) 314, 151-156.). The control GABA response
>0 was substantially potentiated by the positive control drug, chlormethiazole, at a concentration
of 200 uM (Figure 15a). In contrast, a concentration of 200 \M of IVk produced a smaller,
but still significant, potentiation of the control GABA response in the same oocyte (Figure
15a). A decreased efficacy with IVk suggests the possibility that it is a partial allosteric
modulator of GABA A receptor function, and may therefore have more selective behavioural
5 effects than more efficacious compounds.
The effect of a drug that induces brief periods of sedation/hypnosis in animals is
characterized by loss of the righting reflex (loss of the ability of the animal to right its posture
when placed on its back). The "loss of the righting reflex" response is a commonly used test
for sedative-hypnotic agents. IVk produced a dose-dependent and reversible loss of the
righting reflex in mice when given by intraperitoneal injection at doses of 100 and 200 mg/kg
(Figure 15b). Thus, the organic nitrate IVk exhibits the activity of a sedative/hypnotic, and
represents a novel class of compounds that may have utility as new anti-anxiety, sedative
and/ or hypnotic agents.
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